Phenotypic landscape of intestinal organoid regeneration.

The development of intestinal organoids from single adult intestinal stem cells in vitro recapitulates the regenerative capacity of the intestinal epithelium1,2. Here we unravel the mechanisms that orchestrate both organoid formation and the regeneration of intestinal tissue, using an image-based screen to assay an annotated library of compounds. We generate multivariate feature profiles for hundreds of thousands of organoids to quantitatively describe their phenotypic landscape. We then use these phenotypic fingerprints to infer regulatory genetic interactions, establishing a new approach to the mapping of genetic interactions in an emergent system. This allows us to identify genes that regulate cell-fate transitions and maintain the balance between regeneration and homeostasis, unravelling previously unknown roles for several pathways, among them retinoic acid signalling. We then characterize a crucial role for retinoic acid nuclear receptors in controlling exit from the regenerative state and driving enterocyte differentiation. By combining quantitative imaging with RNA sequencing, we show the role of endogenous retinoic acid metabolism in initiating transcriptional programs that guide the cell-fate transitions of intestinal epithelium, and we identify an inhibitor of the retinoid X receptor that improves intestinal regeneration in vivo.

Selective DYRK1A Inhibitor for the Treatment of Type 1 Diabetes: Discovery of 6-Azaindole Derivative GNF2133.

Autoimmune deficiency and destruction in either ß-cell mass or function can cause insufficient insulin levels and, as a result, hyperglycemia and diabetes. Thus, promoting ß-cell proliferation could be one approach toward diabetes intervention. In this report we describe the discovery of a potent and selective DYRK1A inhibitor GNF2133, which was identified through optimization of a 6-azaindole screening hit. In vitro, GNF2133 is able to proliferate both rodent and human ß-cells. In vivo, GNF2133 demonstrated significant dose-dependent glucose disposal capacity and insulin secretion in response to glucose-potentiated arginine-induced insulin secretion (GPAIS) challenge in rat insulin promoter and diphtheria toxin A (RIP-DTA) mice. The work described here provides new avenues to disease altering therapeutic interventions in the treatment of type 1 diabetes (T1D).

Tropifexor-Mediated Abrogation of Steatohepatitis and Fibrosis Is Associated With the Antioxidative Gene Expression Profile in Rodents.

Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid-derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at <1 mg/kg doses was superior to that of OCA at 25 mg/kg in the liver in both NASH models. In a chemical and dietary model of NASH (Stelic animal model [STAM]), tropifexor reversed established fibrosis and reduced the nonalcoholic fatty liver disease activity score and hepatic triglycerides. In an insulin-resistant obese NASH model (amylin liver NASH model [AMLN]), tropifexor markedly reduced steatohepatitis, fibrosis, and profibrogenic gene expression. Transcriptome analysis of livers from AMLN mice revealed 461 differentially expressed genes following tropifexor treatment that included a combination of signatures associated with reduction of oxidative stress, fibrogenesis, and inflammation. Conclusion: Based on preclinical validation in animal models, tropifexor is a promising investigational therapy that is currently under phase 2 development for NASH.

Identification of Potent and Selective RIPK2 Inhibitors for the Treatment of Inflammatory Diseases.

NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.

Pharmacological inhibition of porcupine induces regression of experimental skin fibrosis by targeting Wnt signalling.

OBJECTIVES: Wnt signalling has been implicated in activating a fibrogenic programme in fibroblasts in systemic sclerosis (SSc). Porcupine is an O-acyltransferase required for secretion of Wnt proteins in mammals. Here, we aimed to evaluate the antifibrotic effects of pharmacological inhibition of porcupine in preclinical models of SSc. METHODS: The porcupine inhibitor GNF6231 was evaluated in the mouse models of bleomycin-induced skin fibrosis, in tight-skin-1 mice, in murine sclerodermatous chronic-graft-versus-host disease (cGvHD) and in fibrosis induced by a constitutively active transforming growth factor-ß-receptor I. RESULTS: Treatment with pharmacologically relevant and well-tolerated doses of GNF6231 inhibited the activation of Wnt signalling in fibrotic murine skin. GNF6231 ameliorated skin fibrosis in all four models. Treatment with GNF6231 also reduced pulmonary fibrosis associated with murine cGvHD. Most importantly, GNF6231 prevented progression of fibrosis and showed evidence of reversal of established fibrosis. CONCLUSIONS: These data suggest that targeting the Wnt pathway through inhibition of porcupine provides a potential therapeutic approach to fibrosis in SSc. This is of particular interest, as a close analogue of GNF6231 has already demonstrated robust pathway inhibition in humans and could be available for clinical trials.

Suppressors of Superoxide-H2O2 Production at Site IQ of Mitochondrial Complex I Protect against Stem Cell Hyperplasia and Ischemia-Reperfusion Injury.

Using high-throughput screening we identified small molecules that suppress superoxide and/or H2O2 production during reverse electron transport through mitochondrial respiratory complex I (site IQ) without affecting oxidative phosphorylation (suppressors of site IQ electron leak, "S1QELs"). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site IQ is a normal contributor to mitochondrial superoxide-H2O2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine in vivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H2O2 production by site IQ is involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H2O2 production by site IQ is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site IQ to cell physiology and pathology and have great potential as therapeutic leads.

Inhibition of DYRK1A and GSK3B induces human ß-cell proliferation.

Insufficient pancreatic ß-cell mass or function results in diabetes mellitus. While significant progress has been made in regulating insulin secretion from ß-cells in diabetic patients, no pharmacological agents have been described that increase ß-cell replication in humans. Here we report aminopyrazine compounds that stimulate robust ß-cell proliferation in adult primary islets, most likely as a result of combined inhibition of DYRK1A and GSK3B. Aminopyrazine-treated human islets retain functionality in vitro and after transplantation into diabetic mice. Oral dosing of these compounds in diabetic mice induces ß-cell proliferation, increases ß-cell mass and insulin content, and improves glycaemic control. Biochemical, genetic and cell biology data point to Dyrk1a as the key molecular target. This study supports the feasibility of treating diabetes with an oral therapy to restore ß-cell mass, and highlights a tractable pathway for future drug discovery efforts.

Suppressors of superoxide production from mitochondrial complex III.

Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

A stem cell-based approach to cartilage repair.

Osteoarthritis (OA) is a degenerative joint disease that involves the destruction of articular cartilage and eventually leads to disability. Molecules that promote the selective differentiation of multipotent mesenchymal stem cells (MSCs) into chondrocytes may stimulate the repair of damaged cartilage. Using an image-based high-throughput screen, we identified the small molecule kartogenin, which promotes chondrocyte differentiation (median effective concentration = 100 nM), shows chondroprotective effects in vitro, and is efficacious in two OA animal models. Kartogenin binds filamin A, disrupts its interaction with the transcription factor core-binding factor ß subunit (CBFß), and induces chondrogenesis by regulating the CBFß-RUNX1 transcriptional program. This work provides new insights into the control of chondrogenesis that may ultimately lead to a stem cell-based therapy for osteoarthritis.

Mitochondrial fusion in vitro.

The field of mitochondrial dynamics has received a great deal of attention as a result of a number of studies linking mitochondrial fission and fusion machinery to apoptosis. Specifically, elevated levels of mitochondrial fission or compromised mitochondrial fusion can sensitize cells to apoptotic stimuli. Conversely, stimulation of mitochondrial fusion can render cells resistant to apoptotic stimuli. In addition, the machinery involved in fission and fusion has been spatially linked to Bax, a pro-apoptotic protein. However, the mechanistic implications of interactions between the machinery of mitochondrial fission and fusion and apoptotic effectors are largely unknown. Our understanding of the pathways of mitochondrial fission and fusion have come from genetic studies coupled with direct observation of both fission and fusion components and mitochondrial organelle morphology and behavior in vivo in Saccharomyces cerevisiae. These approaches have identified the key players in both mitochondrial fission and fusion and have generated good models for their roles in mitochondrial dynamics. However, the lack of in vitro systems for studying these processes has impeded a deeper investigation of the mechanism. We have recapitulated the process of mitochondrial fusion in vitro (5). Using this in vitro fusion assay, we have separated outer mitochondrial membrane fusion from inner and identified the mechanistic requirements for each step.

Mitochondrial inner-membrane fusion and crista maintenance requires the dynamin-related GTPase Mgm1.

Mitochondrial outer- and inner-membrane fusion events are coupled in vivo but separable and mechanistically distinct in vitro, indicating that separate fusion machines exist in each membrane. Outer-membrane fusion requires trans interactions of the dynamin-related GTPase Fzo1, GTP hydrolysis, and an intact inner-membrane proton gradient. Inner-membrane fusion also requires GTP hydrolysis but distinctly requires an inner-membrane electrical potential. The protein machinery responsible for inner-membrane fusion is unknown. Here, we show that the conserved intermembrane-space dynamin-related GTPase Mgm1 is required to tether and fuse mitochondrial inner membranes. We observe an additional role of Mgm1 in inner-membrane dynamics, specifically in the maintenance of crista structures. We present evidence that trans Mgm1 interactions on opposing inner membranes function similarly to tether and fuse inner membranes as well as maintain crista structures and propose a model for how the mitochondrial dynamins function to facilitate fusion.

How mitochondria fuse.

Mitochondrial fusion is unique; no paradigm exists to explain how two sets of compositionally distinct membranes become coordinately fused. Genetic approaches coupled with in vivo observations of mitochondrial dynamics and morphology have identified the machinery involved in mitochondrial fusion but these approaches alone yield limited mechanistic insight. The recent recapitulation of mitochondrial fusion in vitro has allowed the fusion process to be dissected into two mechanistically distinct, resolvable steps: outer membrane fusion and inner membrane fusion. Outer membrane fusion requires homotypic trans interactions of the ancient dynamin-related GTPase Fzo1, the proton-gradient component of the inner membrane electrical potential, and low levels of GTP hydrolysis. Fusion of inner membranes requires the electrical component (Deltapsi) of the inner membrane electrical potential and elevated levels of GTP hydrolysis. Regulation of mitochondrial fusion is likely to involve transcript processing in mammalian cells as well as variation in the level of fusion proteins in a given cell; slight changes in the electrical potential of the inner membrane may also serve to fine-tune fusion rates. Mitochondrial fusion components also serve to protect cells against apoptosis through mechanisms that are largely unknown. Resolving the mechanism of mitochondrial fusion will provide insight into the role of fusion components in apoptosis.

Mitochondrial fusion intermediates revealed in vitro.

The events that occur during the fusion of double-membraned mitochondria are unknown. As an essential step toward determining the mechanism of mitochondrial fusion, we have captured this event in vitro. Mitochondrial outer and inner membrane fusion events were separable and mechanistically distinct, but both required guanosine 5'-triphosphate hydrolysis. Homotypic trans interactions of the ancient outer transmembrane guanosine triphosphatase, Fzo1, were required to promote the fusion of mitochondrial outer membranes, whereas electrical potential was also required for fusion of inner membranes. Our conclusions provide fundamental insights into the molecular events driving mitochondrial fusion and advance our understanding of the evolution of mitochondrial fusion in eukaryotic cells.

Evidence for a two membrane-spanning autonomous mitochondrial DNA replisome.

The unit of inheritance for mitochondrial DNA (mtDNA) is a complex nucleoprotein structure termed the nucleoid. The organization of the nucleoid as well as its role in mtDNA replication remain largely unknown. Here, we show in Saccharomyces cerevisiae that at least two populations of nucleoids exist within the same mitochondrion and can be distinguished by their association with a discrete proteinaceous structure that spans the outer and inner mitochondrial membranes. Surprisingly, this two membrane-spanning structure (TMS) persists and self-replicates in the absence of mtDNA. We tested whether TMS functions to direct the replication of mtDNA. By monitoring BrdU incorporation, we observed that actively replicating nucleoids are associated exclusively with TMS. Consistent with TMS's role in mtDNA replication, we found that Mip1, the mtDNA polymerase, is also a stable component of TMS. Taken together, our observations reveal the existence of an autonomous two membrane-spanning mitochondrial replisome as well as provide a mechanism for how mtDNA replication and inheritance may be physically linked.

Staying in aerobic shape: how the structural integrity of mitochondria and mitochondrial DNA is maintained.

The structure and integrity of the mitochondrial compartment are features essential for it to function efficiently. The maintenance of mitochondrial structure in cells ranging from yeast to humans has been shown to require both ongoing fission and fusion. Recent characterization of many of the molecular components that direct mitochondrial fission and fusion events have led to a more complete understanding of how these processes take place. Further, mitochondrial fragmentation observed when cells undergo apoptosis requires mitochondrial fission, underlying the importance of mitochondrial dynamics in cellular homeostasis. Mitochondrial structure also impacts mitochondrial DNA inheritance. Recent studies suggest that faithful transmission of mitochondrial DNA to daughter cells might require a mitochondrial membrane tethering apparatus.