Biology subjects

Kojetin, D.

Publications and source records attributed to Kojetin, D..

12 recordsLinked to original sources

bioRxiv2026

Ileitis abolishes tolerogenic functions of the enterohepatic bile acid pool

Bile acids (BAs) regulate lipid uptake, epithelial integrity, and immune responses in the gut. Hepatocytes synthesize primary BAs, which microbiota metabolize into secondary metabolites. Together, these species form a composite pool that circulates enterohepatically between the liver and intestines. Here, we show that immunoregulatory outputs of the enterohepatic BA pools involve competition between multiple BA species for individual nuclear receptors and are dependent on intestinal health. In healthy mice, the primary BA, tauro {beta} muricholic acid (t{beta}MCA) antagonized ROR{gamma}t-mediated Th17 function in the presence of two secondary BAs (tDCA, tLCA) which activated ROR{gamma}t. Conversely, ileitis in Crohns disease patients and Tnf{delta}ARE mice depleted primary BAs by reducing the number and function of BA transporting enterocytes. In mice, ileitis driven t{beta}MCA depletion enhanced tDCA and tLCA mediated ROR{gamma}t activation and supported Th17 cell function. Replenishing t{beta}MCA in Tnf{delta}ARE mice reestablished BA dependent Th17 cell regulation. Thus, intra pool competition underpins BA immunoregulatory functions and reveals new opportunities for precision Crohns disease therapy.

immunology↗

bioRxiv2025

Shifting the PPARγ conformational ensemble towards a transcriptionally repressive state improves covalent inhibitor efficacy

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates transcription in response to ligand binding at an orthosteric pocket within the ligand-binding domain (LBD). We previously showed that two covalent ligands, T0070907 and GW9662--extensively used as PPAR{gamma} inhibitors to assess off-target activity--weaken but do not completely block ligand binding via an allosteric mechanism associated with pharmacological inverse agonism (Shang et al., 2024). These covalent inhibitors shift the LBD towards a repressive conformation, where the activation function-2 (AF-2) helix 12 occupies the orthosteric pocket, competing with orthosteric ligand binding. Here, we provide additional support for this allosteric mechanism using two covalent inverse agonists, SR33065 and SR36708, which better stabilize the repressive LBD conformation and are more effective inhibitors of--but also do not completely inhibit--ligand cobinding. Furthermore, we show that ligand cobinding can occur with a previously reported PPAR{gamma} dual-site covalent inhibitor, SR16832, which appears to weaken ligand binding through a direct mechanism independent of the allosteric mechanism. These findings underscore the complex nature of the PPAR{gamma} LBD conformational ensemble and highlight the need to develop alternative methods for designing more effective covalent inhibitors.

biochemistry↗

bioRxiv2024

A tethering mechanism underlies Pin1-catalyzed proline cis-trans isomerization at a noncanonical site

The prolyl isomerase Pin1 catalyzes the cis-trans isomerization of proline peptide bonds, a noncovalent post-translational modification that influences cellular and molecular processes, including protein-protein interactions. Pin1 is a two-domain enzyme containing a WW domain that recognizes phosphorylated serine/threonine-proline (pS/pT-P) canonical motifs and an enzymatic PPIase domain that catalyzes proline cis-trans isomerization of pS/pT-P motifs. Here, we show that Pin1 uses a tethering mechanism to bind and catalyze proline cis-trans isomerization of a noncanonical motif in the disordered N-terminal activation function-1 (AF-1) domain of the human nuclear receptor PPAR{gamma}. NMR reveals multiple Pin1 binding regions within the PPAR{gamma} AF-1, including a canonical motif (pS112-P113) that when phosphorylated by the kinase ERK2 binds the Pin1 WW domain with high affinity. NMR methods reveal that Pin1 also binds and accelerates cis-trans isomerization of a noncanonical motif containing a tryptophan-proline motif (W39-P40) previously shown to be involved in an interdomain interaction with the C-terminal ligand-binding domain (LBD) of PPAR{gamma}. Cellular transcription studies combined with mutagenesis and Pin1 inhibitor treatment reveal a functional role for Pin1-mediated acceleration of cis-trans isomerization of the PPAR{gamma} W39-P40 motif. Our data inform a refined model of the Pin1 catalytic mechanism where the WW domain can bind a canonical pS/T-P motif and tether Pin1 to a target, which enables the PPIase domain to exert catalytic cis-trans isomerization at a distal noncanonical site. SIGNIFICANCEPin1 is a multidomain prolyl isomerase enzyme that catalyzes the isomerization of proline peptide bonds, which naturally occur in cis and trans conformations that exchange on a timescale of seconds to minutes, allowing for switch-like effects on target protein structure and function. Previous mechanistic studies using small peptides derived from target substrates revealed Pin1 specifically binds to and displays enzymatic catalysis specificity for substrates containing a phosphorylated serine or threonine followed by a proline (pS/pT-P). Using a large substrate domain from the nuclear receptor peroxisome proliferator activated receptor gamma (PPAR{gamma}), we found that Pin1-catalyzed isomerization can occur at a noncanonical proline distal to a canonical pS/pT-P binding site. Our findings expand the understanding of Pin1-catalyzed enzymatic activities and target substrate functions.

biophysics↗

bioRxiv2024

Unanticipated mechanisms of covalent inhibitor and synthetic ligand cobinding to PPARgamma

Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous lipids and synthetic ligands, including covalent antagonist inhibitors such as GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPAR{gamma} ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPAR{gamma} structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent antagonist inhibitors weaken--but do not prevent--the binding of other synthetic ligands via an allosteric mechanism rather than direct ligand clashing. The covalent ligands shift the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with and reduces the orthosteric binding affinity of non-covalent synthetic ligands. Crystal structures reveal different non-covalent synthetic ligand-specific cobinding mechanisms ranging from alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent ligand binding pose. Our findings not only highlight the significant flexibility of the PPAR{gamma} orthosteric pocket and its ability to accommodate multiple ligands simultaneously, but also demonstrate that GW9662 and T0070907 should not be used as reliable chemical tools to inhibit the binding of other ligands to PPAR{gamma}.

biochemistry↗

bioRxiv2024

Ligand efficacy shifts a nuclear receptor conformational ensemble between transcriptionally active and repressive states

Nuclear receptors (NRs) are thought to dynamically alternate between transcriptionally active and repressive conformations, which are stabilized upon ligand binding. Most NR ligand series exhibit limited bias, primarily consisting of transcriptionally active agonists or neutral antagonists, but not repressive inverse agonists--a limitation that restricts understanding of the functional NR conformational ensemble. Here, we report a NR ligand series for peroxisome proliferator-activated receptor gamma (PPAR{gamma}) that spans a pharmacological spectrum from repression (inverse agonism) to activation (agonism) where subtle structural modifications switch compound activity. While crystal structures provide snapshots of the fully repressive state, NMR spectroscopy and conformation-activity relationship analysis reveals that compounds within the series shift the PPAR{gamma} conformational ensemble between transcriptionally active and repressive conformations that are populated in the apo/ligand-free ensemble. Our findings reveal a molecular framework for minimal chemical modifications that enhance PPAR{gamma} inverse agonism and elucidate their influence on the dynamic PPAR{gamma} conformational ensemble.

biophysics↗

bioRxiv2022

Molecular basis of ligand-dependent Nurr1-RXRα activation

Small molecule compounds that activate transcription of Nurr1-RXR (NR4A2-NR2B1) nuclear receptor heterodimers are implicated in the treatment of neurodegenerative disorders, but function through poorly understood mechanisms. Here, we show that RXR ligands activate Nurr1-RXR through a mechanism that involves ligand-binding domain (LBD) heterodimer proteinprotein interaction (PPI) inhibition, a paradigm distinct from classical pharmacological mechanisms of ligand-dependent nuclear receptor modulation. NMR spectroscopy, protein-protein interaction, cellular transcription assays show that Nurr1-RXR transcriptional activation by RXR ligands is not correlated with classical RXR agonism but instead correlated with weakening Nurr1-RXR LBD heterodimer affinity and heterodimer dissociation. Our data inform a model by which pharmacologically distinct RXR ligands (agonists and Nurr1-RXR selective agonists that function as RXR antagonists) operate as allosteric PPI inhibitors that release a transcriptionally active Nurr1 monomer from a repressive Nurr1-RXR heterodimeric complex. These findings provide a molecular blueprint for ligand activation of Nurr1 transcription via small molecule targeting of Nurr1-RXR.

biochemistry↗

bioRxiv2020

Structural Mechanism Underlying Ligand Binding and Activation of PPARγ

Ligands bind to an occluded orthosteric pocket within the nuclear receptor (NR) ligand-binding domain (LBD). Molecular simulations have revealed several theoretical ligand entry/exit pathways to the orthosteric pocket, but experimentally it remains unclear whether ligand binding proceeds through induced fit or conformational selection mechanisms. Using NMR spectroscopy lineshape analysis, we show that ligand binding to the peroxisome proliferator-activated receptor gamma (PPAR{gamma}) LBD involves a two-step induced fit mechanism including an initial fast step followed by slow conformational change. Surface plasmon resonance and isothermal titration calorimetry heat capacity analysis support the fast kinetic binding step and the conformational change after binding step, respectively. The putative initial ligand binding pose is suggested in several crystal structures of PPAR{gamma} LBD where a ligand is bound to a surface pore formed by helix 3, the {beta}-sheet, and the {Omega}-loop--one of several ligand entry sites suggested in previous targeted and unbiased molecular simulations. These findings, when considered with a recent NMR study showing the activation function-2 (AF-2) helix 12 exchanges in and out of the orthosteric pocket in apo/ligand-free PPAR{gamma}, suggest an activation mechanism whereby agonist binding occurs through an initial encounter complex with the LBD followed by transition of the ligand into the orthosteric pocket concomitant with a conformational change resulting in a solvent-exposed active helix 12 conformation.

biophysics↗

bioRxiv2020

Chemical Systems Biology Reveals Mechanisms of Dissociated Glucocorticoid Receptor Signaling

Glucocorticoids display remarkable anti-inflammatory activity, but their use is limited by on-target adverse effects including insulin resistance and skeletal muscle atrophy. We used a chemical systems biology approach, Ligand Class Analysis (LCA), to examine ligands designed to modulate glucocorticoid receptor activity through distinct structural mechanisms. These ligands displayed diverse activity profiles, providing the variance required to identify target genes and coregulator interactions that were highly predictive of their effects on myocyte glucose disposal and protein balance. Their anti-inflammatory effects were linked to glucose disposal but not muscle atrophy. This approach also predicted selective modulation in vivo, identifying compounds that were muscle sparing or anabolic for protein balance and mitochondrial potential. LCA defined the mechanistic links between the ligand-receptor interface and ligand-driven physiological outcomes, a general approach that can be applied to any ligand-regulated allosteric signaling system.

pharmacology and toxicology↗

bioRxiv2020

Assessment of NR4A Ligands that Directly Bind and Modulate the Orphan Nuclear Receptor Nurr1

ABSTRACTNurr1/NR4A2 is an orphan nuclear receptor transcription factor implicated as a potential drug target for neurological disorders including Alzheimer’s and Parkinson’s diseases. Previous studies identified small molecule modulators of NR4A nuclear receptors including Nurr1 and Nur77/NR4A1; it remains unclear whether these ligands affect Nurr1 through direct binding or indirect non-binding mechanisms. We assessed a panel of twelve ligands reported to affect NR4A activity for Nurr1-dependent and Nurr1-independent transcriptional effects and binding to the Nurr1 ligand-binding domain (LBD). Most of the NR4A ligands show Nurr1-independent effects on transcription in a cell type-specific manner, suggesting they may function through binding to effector proteins whose downstream activities influence Nurr1 function. Protein NMR spectroscopy structural footprinting data show that 4-amino-7-chloroquinoline derivatives (amodiaquine and chloroquine) and cytosporone B directly bind the Nurr1 LBD. In contrast, other NR4A ligands including commercially available compounds such as C-DIM12, celastrol, camptothecin, IP7e, isoalantolactone, and TMPA do not bind the Nurr1 LBD. Interestingly, previous crystal structures indicate that cytosporone B analogs bind to surface pockets in the Nur77 LBD, but protein NMR data indicate cytosporone B likely binds to the Nurr1 orthosteric pocket. These findings should influence medicinal chemistry efforts that desire to optimize Nurr1-binding ligands as opposed to ligands that function through binding to Nurr1 effector proteins.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2019

Quantitative Structural Assessment of Graded Receptor Agonism

Ligand-receptor interactions, which are ubiquitous in physiology, are described by theoretical models of receptor pharmacology. Structural evidence for graded-efficacy receptor conformations predicted by receptor theory has been limited, but is critical to fully validate theoretical models. We applied quantitative structure-function approaches to characterize the effects of structurally similar and structurally diverse agonists on the conformational ensemble of nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR{gamma}). For all ligands, agonist efficacy is correlated to a shift in the conformational ensemble equilibrium from a ground state towards an active state, which is detected by NMR spectroscopy but not observed in crystal structures. For the structurally similar ligands, ligand potency is also correlated to efficacy and conformation, indicating ligand residence times among related analogs can influence receptor conformation and function. Our results derived from quantitative graded activity-conformation correlations provide new experimental evidence and a platform with which to extend and test theoretical models of receptor pharmacology to more accurately describe and predict ligand-dependent receptor activity.

biophysics↗

bioRxiv2018

Structure-function analysis of 1-hydroxypioglitazone, a major in vivo metabolite of the PPARγ agonist pioglitazone

The thiazolidinedione (TZD) pioglitazone (Pio) is an FDA-approved drug for type 2 diabetes mellitus that binds and activates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR{gamma}). Although TZDs have potent antidiabetic effects, they also display harmful side effects that have necessitated a better understanding of their mechanisms of action. In particular, little is known about the effect of in vivo TZD metabolites on the structure and function of PPAR{gamma}. Here, we present a structure-function comparison of Pio and a major in vivo metabolite, 1-hydroxypioglitazone (PioOH). PioOH displayed a lower binding affinity and reduced potency in coregulator recruitment assays compared to Pio. To determine the structural basis of these findings, we solved an X-ray crystal structure of PioOH bound to PPAR{gamma} ligand-binding domain (LBD) and compared it to a published Pio-bound crystal structure. PioOH exhibited an altered hydrogen bonding network that could underlie its reduced affinity and potency compared to Pio. Solution-state structural analysis using NMR spectroscopy and hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PioOH stabilizes the PPAR{gamma} activation function-2 (AF-2) coactivator binding surface better than Pio. In support of AF-2 stabilization, PioOH displayed stabilized coactivator binding in biochemical assays and better transcriptional efficacy (maximal transactivation response) in a cell-based assay that reports on the activity of the PPAR{gamma} LBD. These results, which indicate that Pio hydroxylation affects both its potency and efficacy as a PPAR{gamma} agonist, contribute to our understanding of PPAR{gamma}-binding drug metabolite interactions and may assist in future PPAR{gamma} drug design efforts.

biochemistry↗

bioRxiv2018

A structural mechanism for directing inverse agonism of PPARγ

Small chemical modifications can have significant effects on ligand efficacy and receptor activity, but the underlying structural mechanisms can be difficult to predict from static crystal structures alone. Here we show how a simple phenyl-to-pyridyl substitution between two common covalent orthosteric ligands targeting peroxisome proliferator-activated receptor gamma (PPAR{gamma}) converts a transcriptionally neutral antagonist (GW9662) into an inverse agonist (T0070907). X-ray crystallography, molecular dynamics simulations, and mutagenesis coupled to activity assays reveal a water-mediated hydrogen bond network linking the T0070907 pyridyl group to Arg288 that is essential for inverse agonism. NMR spectroscopy reveals that PPAR{gamma} exchanges between two long-lived conformations when bound to T0070907 but not GW9662, including a conformation that prepopulates a corepressor-bound state, priming PPAR{gamma} for high affinity corepressor binding. Our findings demonstrate that ligand engagement of Arg288 may provide new routes for developing PPAR{gamma} inverse agonist.

biochemistry↗