Characterize direct protein interactions with enrichable, cleavable and latent bioreactive unnatural amino acids.

Latent bioreactive unnatural amino acids (Uaas) have been widely used in the development of covalent drugs and identification of protein interactors, such as proteins, DNA, RNA and carbohydrates. However, it is challenging to perform high-throughput identification of Uaa cross-linking products due to the complexities of protein samples and the data analysis processes. Enrichable Uaas can effectively reduce the complexities of protein samples and simplify data analysis, but few cross-linked peptides were identified from mammalian cell samples with these Uaas. Here we develop an enrichable and multiple amino acids reactive Uaa, eFSY, and demonstrate that eFSY is MS cleavable when eFSY-Lys and eFSY-His are the cross-linking products. An identification software, AixUaa is developed to decipher eFSY mass cleavable data. We systematically identify direct interactomes of Thioredoxin 1 (Trx1) and Selenoprotein M (SELM) with eFSY and AixUaa.

Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells.

Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation. We demonstrated this strategy by identifying direct substrates of thioredoxin in both E. coli and HEK293T cells using two bis-vinyl sulfone chemical cross-linkers BVSB and PDES. We confirmed that BVSB and PDES have high specificity in cross-linking the active site of thioredoxin with its substrates both in vitro and in live cells. Applying live cell cross-linking, we identified 212 putative substrates of thioredoxin in E. coli and 299 putative S-nitrosylation (SNO) substrates of thioredoxin in HEK293T cells. In addition to thioredoxin, we have shown that this strategy can be applied to other proteins in the thioredoxin superfamily. Based on these results, we believe future development of cross-linking techniques will further advance cross-linking mass spectrometry in identifying substrates of other classes of enzymes.

SNX27-driven membrane localisation of OTULIN antagonises linear ubiquitination and NF-κB signalling activation.

BACKGROUND: Linear ubiquitination is a novel type of ubiquitination that plays important physiological roles in signalling pathways such as tumour necrosis factor (TNF) signalling. However, little is known about the regulatory mechanisms of linear ubiquitination, except the well-described enzymatic regulators E3 ligase linear ubiquitin chain assembly complex (LUBAC) and deubiquitinase OTULIN. RESULTS: Previously, we identified SNX27, a member of the sorting nexin family protein, as a selective linear ubiquitin chain interactor in mass spectrometry-based ubiquitin interaction screening. Here, we demonstrated that the interaction between the linear ubiquitin chain and SNX27 is mediated by the OTULIN. Furthermore, we found that SNX27 inhibits LUBAC-mediated linear ubiquitin chain formation and TNFα-induced signalling activation. Mechanistic studies showed that, upon TNFα stimulation, OTULIN-SNX27 is localised to membrane-associated TNF receptor complex, where OTULIN deubiquitinates the linear polyubiquitin chain that formed by the LUBAC complex. Significantly, chemical inhibition of SNX27-retromer translocation by cholera toxin inhibits OTULIN membrane localization. CONCLUSIONS: In conclusion, our study demonstrated that SNX27 inhibits TNFα induced NF-κB signalling activation via facilitating OTULIN to localize to TNF receptor complex.

The natural compound rutaecarpine promotes white adipocyte browning through activation of the AMPK-PRDM16 axis.

The prevalence of obesity is increasing globally and is associated with many metabolic disorders, such as type 2 diabetes and cardiovascular diseases. In recent years, a number of studies suggest that promotion of white adipose browning represents a promising strategy to combat obesity and its related metabolic disorders. The aim of this study was to identify compounds that induce adipocyte browning and elucidate their mechanism of action. Among the 500 natural compounds screened, a small molecule named Rutaecarpine, was identified as a positive regulator of adipocyte browning both in vitro and in vivo. KEGG pathway analysis from RNA-seq data suggested that the AMPK signaling pathway was regulated by Rutaecarpine, which was validated by Western blot analysis. Furthermore, inhibition of AMPK signaling mitigated the browning effect of Rutaecaripine. The effect of Rutaecaripine on adipocyte browning was also abolished upon deletion of Prdm16, a downstream target of AMPK pathway. In collusion, Rutaecarpine is a potent chemical agent to induce adipocyte browning and may serve as a potential drug candidate to treat obesity.

Small molecules targeting RORγt inhibit autoimmune disease by suppressing Th17 cell differentiation.

Th17 cells, a lymphocyte subpopulation that is characterized by the expression of the transcription factor "retinoic acid receptor-related orphan receptor gamma-t" (RORγt), plays an important role in the pathogenesis of autoimmune disease. The current study was set up to discover novel and non-steroidal small-molecule inverse agonists of RORγt and to determine their effects on autoimmune disease. Structure-based virtual screening (SBVS) was used to find compounds targeting RORγt. Flow cytometry was used to detect the Th17 cell differentiation. Inverse agonists were intraperitoneally administered to mice undergoing experimental autoimmune uveitis (EAU), experimental autoimmune encephalomyelitis (EAE) or type 1 diabetes. The effects of the inverse agonists were evaluated by clinical or histopathological scoring. Among 1.3 million compounds screened, CQMU151 and CQMU152 were found to inhibit Th17 cell differentiation without affecting the differentiation of Th1 and Treg lineages (both P = 0.001). These compounds also reduced the severity of EAU (P = 0.01 and 0.013) and functional studies showed that they reduced the number of Th17 cell and the expression of IL-17(Th17), but not IFN-γ(Th1) and TGF-ß(Treg) in mouse retinas. Further studies showed that these compounds may reduce the expression of p-STAT3 by reducing the positive feedback loop of IL-17/IL-6/STAT3. These compounds also reduced the impaired blood-retinal barrier function by upregulating the expression of tight junction proteins. These compounds were also found to reduce the severity of EAE and type 1 diabetes. Our results showed that RORγt inverse agonists may inhibit the development of autoimmune diseases and may provide new clues for the treatment of Th17-mediated immune diseases.

Tbx15 is required for adipocyte browning induced by adrenergic signaling pathway.

OBJECTIVE: The T-box gene Tbx15 is abundantly expressed in adipose tissues, especially subcutaneous and brown fat. Although its expression is correlated with obesity, its precise biological role in adipose tissue is poorly understood in vivo. Here we investigated the function of Tbx15 in brown adipose thermogenesis and white adipose browning in vivo. METHODS: In the present study, we generated adipose-specific Tbx15 knockout (AKO) mice by crossing Tbx15 floxed mice with adiponectin-Cre mice to delineate Tbx15 function in adipose tissues. We systematically investigated the influence of Tbx15 on brown adipose thermogenesis and white adipose browning in mice, as well as the possible underlying molecular mechanism. RESULTS: Upon cold exposure, adipocyte browning in inguinal adipose tissue was significantly impaired in Tbx15 AKO mice. Furthermore, ablation of Tbx15 blocked adipocyte browning induced by ß3 adrenergic agonist CL 316243, which did not appear to alter the expression of Tbx15. Analysis of DNA binding sites using chromatin-immunoprecipitation (ChIP) revealed that TBX15 bound directly to a key region in the Prdm16 promoter, indicating it regulates transcription of Prdm16, the master gene for adipocyte thermogenesis and browning. Compared to control mice, Tbx15 AKO mice displayed increased body weight gain and decreased whole body energy expenditure in response to high fat diets. CONCLUSION: Taken together, these findings suggest that Tbx15 regulates adipocyte browning and might be a potential target for the treatment of obesity.

Linifanib exerts dual anti-obesity effect by regulating adipocyte browning and formation.

Obesity is caused by energy imbalance and accompanied by adipocyte hypertrophy and hyperplasia. Therefore, both enhancement of adipocyte energy expenditure and inhibition of adipogenesis are viable ways to combat obesity. Using the Ucp1-2A-luciferase reporter animal model previously reported by us as a screening platform, a chemical compound Linifanib was identified as a potent inducer of UCP1 expression in primary inguinal adipocytes in vitro and in vivo. Signal pathway analyses showed that Linifanib promoted adipocyte browning by attenuating STAT3 phosphorylation. The effects of Linifanib on adipocyte browning were blocked by the compound, SD19, which activates the STAT3 signaling cascade. Linifanib also inhibited adipocyte differentiation, by blocking mitotic clonal expansion, which could be rescued by STAT3 activator. Taken together, our results indicate that Linifanib might serve as a potential drug for the treatment of obesity.

Small Molecule Derived From Carboxyethylpyrrole Protein Adducts Promotes Angiogenesis in a Mouse Model of Peripheral Arterial Disease.

Background CEP (ω-[2-carboxyethyl]pyrrole) protein adducts are the end products of lipid oxidation associated with inflammation and have been implicated in the induction of angiogenesis in pathological conditions such as tissue ischemia. We synthesized small molecules derived from CEP protein adducts and evaluated the angiogenic effect of the CEP analog CEP 03 in the setting of peripheral arterial disease. Methods and Results The angiogenic effect of CEP 03 was assessed by in vitro analysis of primary human microvascular endothelial cell proliferation and tubelike formation in Matrigel (Corning). In the presence of CEP 03, proliferation of endothelial cells in vitro increased by 27±18% under hypoxic (1% O2) conditions, reaching similar levels to that of VEGF A (vascular endothelial growth factor A) stimulation (22±10%), relative to the vehicle control treatment. A similar effect of CEP 03 was demonstrated in the increased number of tubelike branches in Matrigel, reaching >70% induction in hypoxia, compared with the vehicle control. The therapeutic potential of CEP 03 was further evaluated in a mouse model of peripheral arterial disease by quantification of blood perfusion recovery and capillary density. In the ischemic hind limb, treatment of CEP 03 encapsulated within Matrigel significantly enhanced blood perfusion by 2-fold after 14 days compared with those treated with Matrigel alone. Moreover, these results concurred with histological finding that treatment of CEP 03 in Matrigel resulted in a significant increase in microvessel density compared with Matrigel alone. Conclusions Our data suggest that CEP 03 has a profound positive effect on angiogenesis and neovessel formation and thus has therapeutic potential for treatment of peripheral arterial disease.

Bone-targeted delivery of TGF-ß type 1 receptor inhibitor rescues uncoupled bone remodeling in Camurati-Engelmann disease.

Camurati-Engelmann disease (CED) is a genetic bone-modeling disorder mainly caused by mutations in the gene that encodes transforming growth factor-ß1 (TGF-ß1). Symptoms of CED include bone pain, fractures, and dysplasia. Currently, effective therapies for bone fracture and dysplasia in CED are urgently needed. We have demonstrated that TGF-ß1 is a coupling factor for bone remodeling and is aberrantly activated in CED. Daily injection of TGF-ß type 1 receptor inhibitor (TßR1I) attenuated CED symptoms, but this systemic administration caused serious side effects. In this study, we created a conjugate linking TßR1I and alendronate, which delivered TßR1I specifically to bone. After weekly injection of the conjugate for 8 weeks, normal bone morphology and remodeling in CED mice was maintained with a minimum effective dose 700 times lower than TßR1I injection. Additionally, we found that the conjugate restored normal bone turnover by reducing the number of osteoblasts and osteoclasts, maintained a regular osteogenic microenvironment by regulating the formation of CD31 and Endomucin double-positive vessels, and preserved ordinary bone formation via inhibition of the migration of leptin-receptor-positive cells. Thus, targeting delivery of TßR1I to bone is a promising therapy for CED and other uncoupled bone remodeling disorders.

Spontaneous and specific chemical cross-linking in live cells to capture and identify protein interactions.

Covalently locking interacting proteins in situ is an attractive strategy for addressing the challenge of identifying weak and transient protein interactions, yet it is demanding to execute chemical reactions in live systems in a biocompatible, specific, and autonomous manner. Harnessing proximity-enabled reactivity of an unnatural amino acid incorporated in the bait toward a target residue of unknown proteins, here we genetically encode chemical cross-linkers (GECX) to cross-link interacting proteins spontaneously and selectively in live cells. Obviating an external trigger for reactivity and affording residue specificity, GECX enables the capture of low-affinity protein binding (affibody with Z protein), elusive enzyme-substrate interaction (ubiquitin-conjugating enzyme UBE2D3 with substrate PCNA), and endogenous proteins interacting with thioredoxin in E. coli cells, allowing for mass spectrometric identification of interacting proteins and crosslinking sites. This live cell chemistry-based approach should be valuable for investigating currently intangible protein interactions in vivo for better understanding of biology in physiological settings.

Small molecule Photoregulin3 prevents retinal degeneration in the RhoP23H mouse model of retinitis pigmentosa.

Regulation of rod gene expression has emerged as a potential therapeutic strategy to treat retinal degenerative diseases like retinitis pigmentosa (RP). We previously reported on a small molecule modulator of the rod transcription factor Nr2e3, Photoregulin1 (PR1), that regulates the expression of photoreceptor-specific genes. Although PR1 slows the progression of retinal degeneration in models of RP in vitro, in vivo analyses were not possible with PR1. We now report a structurally unrelated compound, Photoregulin3 (PR3) that also inhibits rod photoreceptor gene expression, potentially though Nr2e3 modulation. To determine the effectiveness of PR3 as a potential therapy for RP, we treated RhoP23H mice with PR3 and assessed retinal structure and function. PR3-treated RhoP23H mice showed significant structural and functional photoreceptor rescue compared with vehicle-treated littermate control mice. These results provide further support that pharmacological modulation of rod gene expression provides a potential strategy for the treatment of RP.

Pharmacological Reprogramming of Somatic Cells for Regenerative Medicine.

Lost or damaged cells in tissues and organs can be replaced by transplanting therapeutically competent cells. This approach requires methods that effectively manipulate cellular identities and properties to generate sufficient numbers of desired cell types for transplantation. These cells can be generated by reprogramming readily available somatic cells, such as fibroblasts, into induced pluripotent stem cells (iPSCs), which can replicate indefinitely and give rise to any somatic cell type. This reprogramming can be achieved with genetic methods, such as forced expression of pluripotency-inducing transcription factors (TFs), which can be further improved, or even avoided, with pharmacological agents. We screened chemical libraries for such agents and identified small molecules that enhance TF-mediated pluripotency induction in somatic cells. We also developed cocktails of small molecules that can functionally replace combinations of TFs required to induce pluripotency in mouse and human somatic cells. Importantly, we devised and established a general strategy to develop effective pharmacological cocktails for specific cellular reprogramming processes. In the search for useful small molecules, we also discovered and characterized previously unknown mechanisms pertinent to cellular reprogramming. A more direct method to access scarce cells for cell transplantation is transdifferentiation, which uses combinations of cell-type specific TFs to reprogram fibroblasts into the target somatic cell types across lineage boundaries. We created an alternative strategy for cellular transdifferentiation that epigenetically activates somatic cells by pairing temporal treatment with reprogramming molecules and tissue-specific signaling molecules to generate cells of multiple lineages. Using this cell-activation and signaling-directed (CASD) transdifferentiation paradigm, we converted fibroblasts into a variety of somatic cells found in major organs, such as the heart, brain, pancreas, and liver. Specifically, we induced, isolated, and expanded (long-term) lineage-specific progenitor cells that can give rise to a defined range of cell types relevant to specific tissues or organs. Transplanting these progenitor cells or their progeny was therapeutically beneficial in animal models of diseases and organ damage. Importantly, we developed chemically defined conditions, without any genetic factors, that convert fibroblasts into cells of the cardiac and neural lineages, further extending the realm of pharmacological reprogramming of cells. Continuously advancing technologies in pharmacological reprogramming of cells may benefit and advance regenerative medicine. The established pharmacological tools have already been applied to enhance the processes of cellular reprogramming and improve the quality of cells for their clinical applications. The rapidly increasing number of readily available bioactive chemical tools will fuel our efforts to reprogram cells for transplantation therapies.

Brown Adipogenic Reprogramming Induced by a Small Molecule.

Brown adipose tissue (BAT) has attracted considerable research interest because of its therapeutic potential to treat obesity and associated metabolic diseases. Augmentation of brown fat mass and/or its function may represent an attractive strategy to enhance energy expenditure. Using high-throughput phenotypic screening to induce brown adipocyte reprogramming in committed myoblasts, we identified a retinoid X receptor (RXR) agonist, bexarotene (Bex), that efficiently converted myoblasts into brown adipocyte-like cells. Bex-treated mice exhibited enlarged BAT mass, enhanced BAT function, and a modest browning effect in subcutaneous white adipose tissue (WAT). Expression analysis showed that Bex initiated several "browning" pathways at an early stage during brown adipocyte reprogramming. Our findings suggest RXRs as new master regulators that control brown and beige fat development and activation, unlike the common adipogenic regulator PPARγ. Moreover, we demonstrated that selective RXR activation may potentially offer a therapeutic approach to manipulate brown/beige fat function in vivo.

Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming.

BACKGROUND: Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells in situ represents a promising strategy for cardiac regeneration. A combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), can convert fibroblasts into induced cardiomyocyte-like cells, albeit with low efficiency in vitro. METHODS: We screened 5500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. RESULTS: We found that a combination of the transforming growth factor-ß inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency 8-fold when added to GMT-overexpressing cardiac fibroblasts. The small molecules also enhanced the speed and quality of cell conversion; we observed beating cells as early as 1 week after reprogramming compared with 6 to 8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared with those exposed to only GMT. Human cardiac reprogramming was similarly enhanced on transforming growth factor-ß and WNT inhibition and was achieved most efficiently with GMT plus myocardin. CONCLUSIONS: Transforming growth factor-ß and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration.

Potential of Small Molecule-Mediated Reprogramming of Rod Photoreceptors to Treat Retinitis Pigmentosa.

Purpose: Mutations in rod photoreceptor genes can cause retinitis pigmentosa (RP). Rod gene expression is regulated by the nuclear hormone receptor, Nr2e3. Genetic deletion of Nr2e3 reprograms rods into cells that resemble cone photoreceptors, and might therefore prevent their death from some forms of RP. There are no identified ligands for Nr2e3; however, reverse agonists might mimic the genetic rescue effect and may be therapeutically useful for the treatment of RP. Methods: We screened for small molecule modulators of Nr2e3 using primary retinal cell cultures and characterized the most potent, which we have named photoregulin1 (PR1), in vitro and in vivo. We also tested the ability of PR1 to slow the progression of photoreceptor degeneration in two common mouse models of autosomal dominant RP, the RhoP23H and the Pde6brd1 mutations. Results: In developing retina, PR1 causes a decrease in rod gene expression and an increase in S opsin+ cones. Photoregulin1 continues to inhibit rod gene expression in adult mice. When applied to two mouse models of RP, PR1 slows the degeneration of photoreceptors. Conclusions: Chemical compounds identified as modulators of Nr2e3 activity may be useful for the treatment of RP through their effects on expression of disease-causing mutant genes.

Human pancreatic beta-like cells converted from fibroblasts.

Pancreatic beta cells are of great interest for biomedical research and regenerative medicine. Here we show the conversion of human fibroblasts towards an endodermal cell fate by employing non-integrative episomal reprogramming factors in combination with specific growth factors and chemical compounds. On initial culture, converted definitive endodermal progenitor cells (cDE cells) are specified into posterior foregut-like progenitor cells (cPF cells). The cPF cells and their derivatives, pancreatic endodermal progenitor cells (cPE cells), can be greatly expanded. A screening approach identified chemical compounds that promote the differentiation and maturation of cPE cells into functional pancreatic beta-like cells (cPB cells) in vitro. Transplanted cPB cells exhibit glucose-stimulated insulin secretion in vivo and protect mice from chemically induced diabetes. In summary, our study has important implications for future strategies aimed at generating high numbers of functional beta cells, which may help restoring normoglycemia in patients suffering from diabetes.

Patient-Specific Induced Pluripotent Stem Cells for Disease Modeling and Phenotypic Drug Discovery.

In vitro cell models are invaluable tools for studying diseases and discovering drugs. Human induced pluripotent stem cells, particularly derived from patients, are an advantageous resource for generating ample supplies of cells to create unique platforms that model disease. This manuscript will review recent developments in modeling a variety of diseases (including their cellular phenotypes) with induced pluripotent stem cells derived from patients. It will also describe how researchers have exploited these models to validate drugs as potential therapeutics for these devastating diseases.

Chemical approaches to cell reprogramming.

Recent advances in cell reprogramming via employing different sets of factors, which allows generation of various cell types that are beyond the downstream developmental lineages from the starting cell type, provide significant opportunities to study fundamental biology and hold enormous promise in regenerative medicine. Small molecules have been identified to enhance and enable reprogramming by regulating various mechanisms, and provide a highly temporal and tunable approach to modulate cellular fate and functions. Here, we review the latest development in cell reprogramming from the perspective of small molecule modulation.

Total syntheses of Tardioxopiperazine A, Isoechinulin A, and Variecolorin C.

First total syntheses of the isoechinulin-type alkaloids: Tardioxopiperazine A, Isoechinulin A, and Variecolorin C have been achieved from a common key intermediate 5, which was derived from a regiocontrolled Stille cross-coupling reaction of an allylindium reagent.

Concise total syntheses of (+)-strictifolione and (6R)-6-[(4R,6R)-4,6-dihydroxy-10-phenyldec-1-enyl]-5,6-dihydro-2H-pyran-2-one.

Concise and efficient asymmetric total syntheses of (+)-strictifolione 1 and (6R)-6-[(4R,6R)-4,6-dihydroxy-10-phenyldec-1-enyl]-5,6-dihydro-2H-pyran-2-one 2 have been achieved based on the strategic application of one-pot double allylboration and ring-closing metathesis reactions. The total syntheses proceeded in only five and seven steps, respectively, from readily available 3-butenal and represent the shortest syntheses of 1 and 2 reported to date.