ABSTRACT
The salt-inducible kinases (SIK) 1-3 are key regulators of pro- versus anti-inflammatory cytokine responses during innate immune activation. The lack of highly SIK-family or SIK isoform-selective inhibitors suitable for repeat, oral dosing has limited the study of the optimal SIK isoform selectivity profile for suppressing inflammation in vivo. To overcome this challenge, we devised a structure-based design strategy for developing potent SIK inhibitors that are highly selective against other kinases by engaging two differentiating features of the SIK catalytic site. This effort resulted in SIK1/2-selective probes that inhibit key intracellular proximal signaling events including reducing phosphorylation of the SIK substrate cAMP response element binding protein (CREB) regulated transcription coactivator 3 (CRTC3) as detected with an internally generated phospho-Ser329-CRTC3-specific antibody. These inhibitors also suppress production of pro-inflammatory cytokines while inducing anti-inflammatory interleukin-10 in activated human and murine myeloid cells and in mice following a lipopolysaccharide challenge. Oral dosing of these compounds ameliorates disease in a murine colitis model. These findings define an approach to generate highly selective SIK1/2 inhibitors and establish that targeting these isoforms may be a useful strategy to suppress pathological inflammation.
Subject(s)
Cyclic AMP Response Element-Binding Protein , Protein Serine-Threonine Kinases , Mice , Humans , Animals , Protein Serine-Threonine Kinases/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cytokines , Inflammation/drug therapy , Protein Isoforms , Anti-Inflammatory Agents/pharmacology , Immunity, Innate , Transcription FactorsABSTRACT
The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here, we demonstrated that salt-inducible kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole-tissue and single-cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Finally, in a podocyte injury model of chronic kidney disease-mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful for stimulation of 1,25-vitamin D production in CKD-MBD.
Subject(s)
Chronic Kidney Disease-Mineral and Bone Disorder , Renal Insufficiency, Chronic , Mice , Humans , Animals , Vitamin D/metabolism , Parathyroid Hormone/genetics , Parathyroid Hormone/metabolism , Calcium/metabolism , 25-Hydroxyvitamin D3 1-alpha-Hydroxylase/metabolism , Chronic Kidney Disease-Mineral and Bone Disorder/metabolism , Kidney/metabolism , Renal Insufficiency, Chronic/metabolism , Homeostasis , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
Bone formation and resorption are typically coupled, such that the efficacy of anabolic osteoporosis treatments may be limited by bone destruction. The multi-kinase inhibitor YKL-05-099 potently inhibits salt inducible kinases (SIKs) and may represent a promising new class of bone anabolic agents. Here, we report that YKL-05-099 increases bone formation in hypogonadal female mice without increasing bone resorption. Postnatal mice with inducible, global deletion of SIK2 and SIK3 show increased bone mass, increased bone formation, and, distinct from the effects of YKL-05-099, increased bone resorption. No cell-intrinsic role of SIKs in osteoclasts was noted. In addition to blocking SIKs, YKL-05-099 also binds and inhibits CSF1R, the receptor for the osteoclastogenic cytokine M-CSF. Modeling reveals that YKL-05-099 binds to SIK2 and CSF1R in a similar manner. Dual targeting of SIK2/3 and CSF1R induces bone formation without concomitantly increasing bone resorption and thereby may overcome limitations of most current anabolic osteoporosis therapies.
Subject(s)
Bone Resorption/genetics , Osteogenesis/genetics , Protein Serine-Threonine Kinases/genetics , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Animals , Female , Male , Mice , Protein Serine-Threonine Kinases/metabolism , Random Allocation , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolismABSTRACT
Immune health requires innate and adaptive immune cells to engage precisely balanced pro- and anti-inflammatory forces. We employ the concept of chemical immunophenotypes to classify small molecules functionally or mechanistically according to their patterns of effects on primary innate and adaptive immune cells. The high-specificity, low-toxicity cyclin-dependent kinase 8 (CDK8) inhibitor 16-didehydro-cortistatin A (DCA) exerts a distinct tolerogenic profile in both innate and adaptive immune cells. DCA promotes regulatory T cells (Treg) and Th2 differentiation while inhibiting Th1 and Th17 differentiation in both murine and human cells. This unique chemical immunophenotype led to mechanistic studies showing that DCA promotes Treg differentiation in part by regulating a previously undescribed CDK8-GATA3-FOXP3 pathway that regulates early pathways of Foxp3 expression. These results highlight previously unappreciated links between Treg and Th2 differentiation and extend our understanding of the transcription factors that regulate Treg differentiation and their temporal sequencing. These findings have significant implications for future mechanistic and translational studies of CDK8 and CDK8 inhibitors.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Forkhead Transcription Factors/metabolism , GATA3 Transcription Factor/metabolism , Heterocyclic Compounds, 4 or More Rings/pharmacology , Immune Tolerance/drug effects , Immunophenotyping , Isoquinolines/pharmacology , Adolescent , Adult , Animals , CD4-Positive T-Lymphocytes/drug effects , Cell Differentiation/drug effects , Cyclin-Dependent Kinase 8/metabolism , Humans , Immunity, Innate/drug effects , Mice, Inbred BALB C , Middle Aged , Phosphorylation/drug effects , Proto-Oncogene Proteins c-jun/metabolism , STAT Transcription Factors/metabolism , Signal Transduction/drug effects , Young AdultABSTRACT
Enhancing production of the anti-inflammatory cytokine interleukin-10 (IL-10) is a promising strategy to suppress pathogenic inflammation. To identify new mechanisms regulating IL-10 production, we conducted a phenotypic screen for small molecules that enhance IL-10 secretion from activated dendritic cells. Mechanism-of-action studies using a prioritized hit from the screen, BRD6989, identified the Mediator-associated kinase CDK8, and its paralog CDK19, as negative regulators of IL-10 production during innate immune activation. The ability of BRD6989 to upregulate IL-10 is recapitulated by multiple, structurally differentiated CDK8 and CDK19 inhibitors and requires an intact cyclin C-CDK8 complex. Using a highly parallel pathway reporter assay, we identified a role for enhanced AP-1 activity in IL-10 potentiation following CDK8 and CDK19 inhibition, an effect associated with reduced phosphorylation of a negative regulatory site on c-Jun. These findings identify a function for CDK8 and CDK19 in regulating innate immune activation and suggest that these kinases may warrant consideration as therapeutic targets for inflammatory disorders.
Subject(s)
Cyclin-Dependent Kinase 8/metabolism , Interleukin-10/biosynthesis , Myeloid Cells/drug effects , Small Molecule Libraries/pharmacology , Animals , Cells, Cultured , Cyclin-Dependent Kinase 8/immunology , Dose-Response Relationship, Drug , Humans , Interleukin-10/immunology , Mice , Mice, Inbred C57BL , Molecular Structure , Myeloid Cells/immunology , Myeloid Cells/metabolism , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
Parathyroid hormone (PTH) activates receptors on osteocytes to orchestrate bone formation and resorption. Here we show that PTH inhibition of SOST (sclerostin), a WNT antagonist, requires HDAC4 and HDAC5, whereas PTH stimulation of RANKL, a stimulator of bone resorption, requires CRTC2. Salt inducible kinases (SIKs) control subcellular localization of HDAC4/5 and CRTC2. PTH regulates both HDAC4/5 and CRTC2 localization via phosphorylation and inhibition of SIK2. Like PTH, new small molecule SIK inhibitors cause decreased phosphorylation and increased nuclear translocation of HDAC4/5 and CRTC2. SIK inhibition mimics many of the effects of PTH in osteocytes as assessed by RNA-seq in cultured osteocytes and following in vivo administration. Once daily treatment with the small molecule SIK inhibitor YKL-05-099 increases bone formation and bone mass. Therefore, a major arm of PTH signalling in osteocytes involves SIK inhibition, and small molecule SIK inhibitors may be applied therapeutically to mimic skeletal effects of PTH.
Subject(s)
Bone and Bones/drug effects , Osteocytes/drug effects , Osteogenesis/drug effects , Parathyroid Hormone/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Active Transport, Cell Nucleus/drug effects , Adaptor Proteins, Signal Transducing , Animals , Bone and Bones/cytology , Bone and Bones/metabolism , Gene Expression Regulation , Glycoproteins/antagonists & inhibitors , Glycoproteins/genetics , Glycoproteins/metabolism , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Humans , Intercellular Signaling Peptides and Proteins , Mice , Mice, Knockout , Osteocytes/cytology , Osteocytes/metabolism , Osteogenesis/genetics , Parathyroid Hormone/metabolism , Phosphorylation/drug effects , Primary Cell Culture , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , RANK Ligand/antagonists & inhibitors , RANK Ligand/genetics , RANK Ligand/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Salt-inducible kinases (SIKs) are promising therapeutic targets for modulating cytokine responses during innate immune activation. The study of SIK inhibition in animal models of disease has been limited by the lack of selective small-molecule probes suitable for modulating SIK function in vivo. We used the pan-SIK inhibitor HG-9-91-01 as a starting point to develop improved analogs, yielding a novel probe 5 (YKL-05-099) that displays increased selectivity for SIKs versus other kinases and enhanced pharmacokinetic properties. Well-tolerated doses of YKL-05-099 achieve free serum concentrations above its IC50 for SIK2 inhibition for >16 h and reduce phosphorylation of a known SIK substrate in vivo. While in vivo active doses of YKL-05-099 recapitulate the effects of SIK inhibition on inflammatory cytokine responses, they did not induce metabolic abnormalities observed in Sik2 knockout mice. These results identify YKL-05-099 as a useful probe to investigate SIK function in vivo and further support the development of SIK inhibitors for treatment of inflammatory disorders.
Subject(s)
Molecular Probes/chemistry , Protein Serine-Threonine Kinases/metabolism , Animals , Cells, Cultured , Inhibitory Concentration 50 , Mice , Mice, Knockout , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistryABSTRACT
Reporter gene assays are a venerable tool for studying signaling pathways, but they lack the throughput and complexity necessary to contribute to a systems-level understanding of endogenous signaling networks. We present a parallel reporter assay, transcription factor activity sequencing (TF-seq), built on synthetic DNA enhancer elements, which enables parallel measurements in primary cells of the transcriptome and transcription factor activity from more than 40 signaling pathways. Using TF-seq in Myd88(-/-) macrophages, we captured dynamic pathway activity changes underpinning the global transcriptional changes of the innate immune response. We also applied TF-seq to investigate small molecule mechanisms of action and find a role for NF-κB activation and coordination of the STAT1 response in the macrophage reaction to the anti-inflammatory natural product halofuginone. Simultaneous TF-seq and global gene expression profiling represent an integrative approach for gaining mechanistic insight into pathway activity and transcriptional changes that result from genetic and small molecule perturbations.
Subject(s)
Sequence Analysis, RNA , Base Sequence , Gene Expression Profiling , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , NF-kappa B , RNA , TranscriptomeABSTRACT
Androgen receptor (AR)-dependent transcription is a major driver of prostate tumor cell proliferation. Consequently, it is the target of several antitumor chemotherapeutic agents, including the AR antagonist MDV3100/enzalutamide. Recent studies have shown that a single AR mutation (F876L) converts MDV3100 action from an antagonist to an agonist. Here we describe the generation of a novel class of selective androgen receptor degraders (SARDs) to address this resistance mechanism. Molecules containing hydrophobic degrons linked to small-molecule AR ligands induce AR degradation, reduce expression of AR target genes and inhibit proliferation in androgen-dependent prostate cancer cell lines. These results suggest that selective AR degradation may be an effective therapeutic prostate tumor strategy in the context of AR mutations that confer resistance to second-generation AR antagonists.
Subject(s)
Androgen Receptor Antagonists/chemistry , Androgen Receptor Antagonists/pharmacology , Proteolysis/drug effects , Receptors, Androgen/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Benzamides , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Humans , Hydrophobic and Hydrophilic Interactions , Male , Nitriles , Phenylthiohydantoin/analogs & derivatives , Phenylthiohydantoin/chemistry , Phenylthiohydantoin/pharmacology , Point Mutation , Prostate/drug effects , Prostate/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Receptors, Androgen/geneticsABSTRACT
The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity.
Subject(s)
Cell Differentiation/immunology , Homeostasis/immunology , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/metabolism , Animals , Cell Culture Techniques , Harmine/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Dyrk KinasesABSTRACT
Manipulating cytokine function with protein-based drugs has proven effective for treating a wide variety of autoimmune and autoinflammatory disorders. However, the limited ability of protein-based drugs to modulate intracellular targets, including many implicated by studies of the genetics and physiology of these diseases, and to coordinately neutralize redundant inflammatory cytokines, suggests an important and complementary role for small molecules in immunomodulatory drug development. The recent clinical approval of Janus kinase and phosphodiesterase inhibitors, along with emerging evidence from other compound classes, firmly establish small molecules as effective tools for modulating therapeutically relevant proteins that give rise to aberrant cytokine signaling or mediate its downstream consequences.
Subject(s)
Autoimmune Diseases/drug therapy , Autoimmune Diseases/immunology , Cytokines/immunology , Immunologic Factors/pharmacology , Small Molecule Libraries/pharmacology , Animals , Drug Discovery , HumansABSTRACT
Genetic alterations that reduce the function of the immunoregulatory cytokine IL-10 contribute to colitis in mouse and man. Myeloid cells such as macrophages (MΦs) and dendritic cells (DCs) play an essential role in determining the relative abundance of IL-10 versus inflammatory cytokines in the gut. As such, using small molecules to boost IL-10 production by DCs-MΦs represents a promising approach to increase levels of this cytokine specifically in gut tissues. Toward this end, we screened a library of well-annotated kinase inhibitors for compounds that enhance production of IL-10 by murine bone-marrow-derived DCs stimulated with the yeast cell wall preparation zymosan. This approach identified a number of kinase inhibitors that robustly up-regulate IL-10 production including the Food and Drug Administration (FDA)-approved drugs dasatinib, bosutinib, and saracatinib that target ABL, SRC-family, and numerous other kinases. Correlating the kinase selectivity profiles of the active compounds with their effect on IL-10 production suggests that inhibition of salt-inducible kinases (SIKs) mediates the observed IL-10 increase. This was confirmed using the SIK-targeting inhibitor HG-9-91-01 and a series of structural analogs. The stimulatory effect of SIK inhibition on IL-10 is also associated with decreased production of the proinflammatory cytokines IL-1ß, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in human DCs-MΦs and anti-inflammatory CD11c(+) CX3CR1(hi) cells isolated from murine gut tissue. Collectively, these studies demonstrate that SIK inhibition promotes an anti-inflammatory phenotype in activated myeloid cells marked by robust IL-10 production and establish these effects as a previously unidentified activity associated with several FDA-approved multikinase inhibitors.
Subject(s)
Dendritic Cells/drug effects , Dendritic Cells/immunology , Interleukin-10/biosynthesis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Aniline Compounds/pharmacology , Animals , Cells, Cultured , Cyclic AMP Response Element-Binding Protein/metabolism , Cytokines/biosynthesis , Dasatinib , Dendritic Cells/enzymology , Drug Evaluation, Preclinical , Humans , Inflammation Mediators/metabolism , Inflammatory Bowel Diseases/drug therapy , Inflammatory Bowel Diseases/enzymology , Inflammatory Bowel Diseases/immunology , Intestine, Small/drug effects , Intestine, Small/enzymology , Intestine, Small/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Cells/drug effects , Myeloid Cells/enzymology , Myeloid Cells/immunology , Nitriles/pharmacology , Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacology , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemistry , Pyrimidines/pharmacology , Quinolines/pharmacology , Signal Transduction/drug effects , Signal Transduction/immunology , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/enzymology , T-Lymphocytes, Regulatory/immunology , Thiazoles/pharmacology , Transcription Factors/metabolismABSTRACT
New HyTs are a knockout: we previously reported that labeling HaloTag proteins with low molecular weight hydrophobic tags (HyTs) leads to targeted degradation of HaloTag fusion proteins. In this report, we employed a chemical approach to extend this hydrophobic tagging methodology to highly stabilized proteins by synthesizing and evaluating a library of HyTs, which led to the identification of HyT36.
Subject(s)
Recombinant Fusion Proteins/metabolism , HEK293 Cells , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Structure , Recombinant Fusion Proteins/chemistryABSTRACT
Identification of methionine aminopeptidase-2 (MetAP-2) as the molecular target of the antiangiogenic compound TNP-470 has sparked interest in N-terminal Met excision's (NME) role in endothelial cell biology. In this regard, we recently demonstrated that MetAP-2 inhibition suppresses Wnt planar cell polarity (PCP) signaling and that endothelial cells depend on this pathway for normal function. Despite this advance, the substrate(s) whose activity is altered upon MetAP-2 inhibition, resulting in loss of Wnt PCP signaling, is not known. Here we identify the small G protein Rab37 as a MetAP-2-specific substrate that accumulates in the presence of TNP-470. A functional role for aberrant Rab37 accumulation in TNP-470's mode of action is demonstrated using a Rab37 point mutant that is resistant to NME, because expression of this mutant phenocopies the effects of MetAP-2 inhibition on Wnt PCP signaling-dependent processes.
Subject(s)
Aminopeptidases/metabolism , Cell Polarity , Metalloendopeptidases/metabolism , Wnt Signaling Pathway , rab GTP-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Aminopeptidases/genetics , Angiogenesis Inhibitors/pharmacology , Animals , Base Sequence , Cell Proliferation , Cyclohexanes/pharmacology , Dishevelled Proteins , Embryo, Nonmammalian , Fatty Acids, Unsaturated/pharmacology , Gene Knockdown Techniques , Growth Inhibitors/metabolism , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Metalloendopeptidases/genetics , Molecular Sequence Data , Mutation , O-(Chloroacetylcarbamoyl)fumagillol , Phosphoproteins/genetics , Phosphoproteins/metabolism , Sesquiterpenes/pharmacology , Zebrafish/embryology , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , rab GTP-Binding Proteins/geneticsABSTRACT
The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface. Hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated and transmembrane HaloTag fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1(G12V)-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small-molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models.
Subject(s)
Biosensing Techniques/methods , Fluorescent Dyes/chemistry , Animals , Cell Line , Humans , Hydrophobic and Hydrophilic Interactions , Luminescent Proteins/chemistry , Mice , Molecular Structure , Recombinant Proteins , Sensitivity and Specificity , Staining and Labeling , ZebrafishABSTRACT
Cells generate adenosine triphosphate (ATP) by glycolysis and by oxidative phosphorylation (OXPHOS). Despite the importance of having sufficient ATP available for the energy-dependent processes involved in immune activation, little is known about the metabolic adaptations that occur in vivo to meet the increased demand for ATP in activated and proliferating lymphocytes. We found that bone marrow (BM) cells proliferating after BM transplantation (BMT) increased aerobic glycolysis but not OXPHOS, whereas T cells proliferating in response to alloantigens during graft-versus-host disease (GVHD) increased both aerobic glycolysis and OXPHOS. Metabolomic analysis of alloreactive T cells showed an accumulation of acylcarnitines consistent with changes in fatty acid oxidation. Alloreactive T cells also exhibited a hyperpolarized mitochondrial membrane potential (ΔΨm), increased superoxide production, and decreased amounts of antioxidants, whereas proliferating BM cells did not. Bz-423, a small-molecule inhibitor of the mitochondrial F(1)F(0) adenosine triphosphate synthase (F(1)F(0)-ATPase), selectively increased superoxide and induced the apoptosis of alloreactive T cells, which arrested established GVHD in several BMT models without affecting hematopoietic engraftment or lymphocyte reconstitution. These findings challenge the current paradigm that activated T cells meet their increased demands for ATP through aerobic glycolysis, and identify the possibility that bioenergetic and redox characteristics can be selectively exploited as a therapeutic strategy for immune disorders.
Subject(s)
Apoptosis/immunology , Graft vs Host Disease/immunology , Isoantigens/immunology , Oxidative Phosphorylation , T-Lymphocytes/metabolism , Animals , Apoptosis/drug effects , Benzodiazepines/pharmacology , Benzodiazepines/therapeutic use , Bone Marrow Cells/metabolism , Bone Marrow Transplantation/immunology , Female , Graft vs Host Disease/drug therapy , Lactates/metabolism , Lymphocyte Activation , Metabolome , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mitochondrial Proton-Translocating ATPases/antagonists & inhibitors , Mitochondrial Proton-Translocating ATPases/metabolism , Oxygen Consumption , Reactive Oxygen Species/metabolism , T-Lymphocytes/drug effectsABSTRACT
7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(napthalen-2-ylmetyl)-4,5,-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423) is a proapoptotic 1,4-benzodiazepine that potently suppresses disease in the murine model of lupus by selectively killing pathogenic lymphocytes. In MRL/MpJ-Fas(lpr) (MRL-lpr) mice, Bz-423 overcomes deficient expression of the Fas death receptor and hyperactivation of antiapoptotic phosphatidylinositol 3-kinase (PI3K)-Akt signaling to specifically kill pathogenic CD4(+) T cells. Bz-423 binds to the oligomycin-sensitivity-conferring protein component of the mitochondrial F(0)F(1)-ATPase, which modulates the enzyme leading to formation of superoxide by the mitochondrial respiratory chain. Scavenging this reactive oxygen species blocks all subsequent components of the apoptotic cascade. To gain insight into how apoptotic signaling activated by Bz-423-induced superoxide contributes to the selective depletion of MRL-lpr CD4(+) T cells, we characterized the death mechanism in a CD4(+) T cell leukemia line (Jurkat). Although Bz-423-induced superoxide indirectly inactivates Akt, this response is not required for T cell death. Apoptosis instead results from parallel increases in levels of the proapoptotic Bcl-2 proteins Noxa and Bak leading to specific activation of Bak, mitochondrial outer membrane permeabilization, and a commitment to apoptosis. By directly up-regulating proteins that trigger loss of mitochondrial outer membrane integrity, Bz-423 bypasses defective Fas function and antiapoptotic PI3K-Akt signaling in MRL-lpr CD4(+) T cells. Moreover, because disease-associated abnormalities should sensitize autoreactive CD4(+) T cells to transcriptional up-regulation of Noxa by redox signals and to Bak-dependent apoptosis, the apoptotic mechanism elucidated in Jurkat cells provides important clues into the cell-type- and disease-selective effects of Bz-423 in MRL-lpr mice.
Subject(s)
Apoptosis/physiology , Autoimmunity/immunology , Benzodiazepines/pharmacology , Lymphocytes/immunology , Proton-Translocating ATPases/physiology , Signal Transduction/physiology , Adenosine Triphosphate/metabolism , Animals , Apoptosis/drug effects , Blotting, Western , CD4-Positive T-Lymphocytes/drug effects , Cell Death/drug effects , Cell Survival , DNA/biosynthesis , DNA/genetics , Humans , Jurkat Cells , MAP Kinase Kinase 4/genetics , Mice , Microscopy, Fluorescence , Oncogene Protein v-akt/genetics , RNA, Small Interfering , Reactive Oxygen Species/metabolism , Transfection , bcl-2 Homologous Antagonist-Killer Protein/metabolismABSTRACT
7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(naphthalen-2-ylmethyl)-4,5-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423) is a benzodiazepine that has cytotoxic and cytostatic activity against a variety of cells in vivo and in vitro. In the present study, we demonstrate that Bz-423 (formulated for topical delivery) reduces epidermal hyperplasia in human psoriatic skin after transplantation to severe, combined immunodeficient (scid) mice. Bz-423 also suppresses the hyperplasia that develops in nonpsoriatic human skin as a consequence of transplantation to scid mice. Proliferation of human epidermal keratinocytes in monolayer culture was suppressed by Bz-423 at concentrations of 0.5 to 2.0 muM (noncytotoxic concentrations). Keratinocyte growth inhibition was accompanied by increased oxidant generation in Bz-423-treated cells, and treatment with vitamin E along with Bz-423 reversed the growth inhibition. Growth inhibition was accompanied by a redistribution of beta-catenin from a cytoplasmic pool to the cell membrane and by reduced levels of c-myc and cyclin D1 (two molecules associated with Wnt pathway signaling). Several analogs of Bz-423 were examined for antiproliferative activity against human epidermal keratinocytes and human dermal fibroblasts in monolayer culture. Each of the analogs tested suppressed growth of both cell types, but in all cases, keratinocytes were more sensitive than fibroblasts. Two of the compounds were found to suppress epidermal hyperplasia induced with all-trans retinoic acid in organ cultures of human skin. Taken together, these data show that Bz-423 and certain analogs produce biological responses in skin cells in vitro and in vivo that are consistent with therapeutic goals for treating psoriasis or epidermal hyperplasia resulting from other causes.
Subject(s)
Benzodiazepines/therapeutic use , Disease Models, Animal , Keratinocytes/cytology , Psoriasis/drug therapy , Severe Combined Immunodeficiency/drug therapy , Skin Transplantation , Animals , Benzodiazepines/pharmacology , Cell Proliferation/drug effects , Humans , Keratinocytes/drug effects , Mice , Mice, SCID , Psoriasis/pathology , Severe Combined Immunodeficiency/pathology , Skin Transplantation/immunology , Skin Transplantation/methodsABSTRACT
A family of 1,4-benzodiazepine-2,5-diones (BZDs) has been synthesized and evaluated against transformed B- and T-cells for lymphotoxic members. A large aromatic group on the C3 position is critical for cytotoxicity. When the C3 moiety contains an electron-rich heterocycle, the resulting BZDs have sub-micromolar potency and are selective for T-cells. Cell death is consistent with apoptosis and does not result from inhibition of the mitochondrial F(o)F1-ATPase, which is the molecular target of recently reported cytotoxic 1,4-benzodiazepines. Collectively, these studies begin to characterize some of the structural elements required for the activity of a novel family of T-cell-selective lymphotoxic agents.