Thomas B. Sundberg

Small-molecule hydrophobic tagging–induced degradation of HaloTag fusion proteins

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 that bind a bacterial dehalogenase (HaloTag protein) and present a hydrophobic group on its surface. Remarkably, hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated, and transmembrane fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting RasG12V-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.

Manipulating the Bioenergetics of Alloreactive T Cells Causes Their Selective Apoptosis and Arrests Graft-Versus-Host Disease

Bioenergetic properties differentiate alloreactive T cells from other proliferating cells and can be exploited to arrest GVHD in mice. A Xenophobe’s Guide to Treating Graft-Versus-Host Disease Immune cells are in essence xenophobes—they distinguish and then attack cells that are foreign to the body. This prejudice is helpful in the context of infection or cancer; the immune system raises destructive responses directed against stranger cells that have been infected or transformed while remaining calm in the presence of healthy cells recognized as “self.” However, when immune cells are transferred to a new host, for example, by bone marrow transplantation, these cells see the patient’s own tissues as foreign and attack, resulting in graft-versus-host disease (GVHD). Gatza et al. have found a way to differentiate these so-called alloreactive donor cells from non-alloreactive donor cells and host immune cells and thus provide a target for preventing GVHD. Cells can generate energy in the form of adenosine triphosphate (ATP) through either aerobic glycolysis or oxidative phosphorylation (OXPHOS). In general, proliferating lymphocytes preferentially use aerobic glycolysis, which produces significantly fewer ATP molecules than OXPHOS, but little is known about the specific metabolic requirements of proliferating alloreactive T cells. Gatza et al. found that after bone marrow transplantation, alloreactive T cells, but not other proliferating T cells and bone marrow cells, up-regulated both aerobic glycolysis and OXPHOS to meet their increased energy demand. Relative to other proliferating cells, alloreactive T cells produced higher concentrations of acylcarnitines—fatty acid oxidation intermediates that transport fatty acids into the mitochondrial matrix. Further highlighting their altered metabolism, alloreactive T cells could be specifically killed by Bz-423, a small-molecule inhibitor of the mitochondrial F1F0-ATPase, and Bz-423 decreased the severity of GVHD in mice without impairing transplant engraftment and bone marrow reconstitution. Thus, bioenergetic differences may provide targets for order-maintaining therapeutics that stop alloreactive T cells from attacking perceived foreigners in the transplanted immune cells’ new neighborhood. 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 F1F0 adenosine triphosphate synthase (F1F0-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.

Identification of hydrophobic tags for the degradation of stabilized proteins.

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.

The kinase DYRK1A reciprocally regulates the differentiation of Th17 and regulatory T cells

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. DOI: http://dx.doi.org/10.7554/eLife.05920.001

Small‐Molecule‐Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging

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.

Disruption of Wnt Planar Cell Polarity Signaling by Aberrant Accumulation of the MetAP-2 Substrate Rab37

Identification of methionine aminopeptidase-2 (MetAP-2) as the molecular target of the antiangiogenic compound TNP-470 has sparked interest in N-terminal Met excisions (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-470s 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.

SIKs control osteocyte responses to parathyroid hormone

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.

Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells

Significance IL-10 plays an essential role in maintaining gut immune homeostasis as evidenced by the link between genetic perturbation of this anti-inflammatory cytokine and inflammatory bowel disease (IBD). Here, we describe a small-molecule screen that identified inhibition of salt-inducible kinases (SIKs) as a strategy to enhance IL-10 production by macrophages and dendritic cells. Significantly, the IL-10–potentiating effects of SIK inhibition are associated with reduced secretion of the inflammatory cytokines IL-1β, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in cells relevant to IBD including anti-inflammatory CD11c+ CX3CR1hi cells from murine gut tissue and in human dendritic cells and macrophages. Collectively, these results identify SIK inhibition as a promising approach to treat IBD by increasing gut IL-10 levels. 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-1β, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in human DCs–MΦs and anti-inflammatory CD11c+ CX3CR1hi 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.

Development of Chemical Probes for Investigation of Salt-Inducible Kinase Function in Vivo.

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.

Small-molecule control of cytokine function: new opportunities for treating immune disorders

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.