SZN-413, a FZD4 Agonist, as a Potential Novel Therapeutic for the Treatment of Diabetic Retinopathy.

Purpose: There remains a high unmet need for therapies with new mechanisms of action to achieve reperfusion of ischemic retina in diabetic retinopathy. We examined whether a novel frizzled class receptor 4 (FZD4) agonist could promote regeneration of functional blood vessels in animal models of retinopathy. Methods: We developed a novel Norrin mimetic (SZN-413-p) targeting FZD4 and low-density lipoprotein receptor-related protein 5 (LRP5) and examined its effect on retinal and brain endothelial cells in vitro. SZN-413-p was subsequently humanized, resulting in the therapeutic candidate SZN-413, and was examined in animal models of retinopathy. In an oxygen-induced retinopathy mouse model, avascular and neovascularization areas were measured. Furthermore, in a vascular endothelial growth factor (VEGF)-induced retinal vascular leakage rabbit model, the impact on vascular leakage by SZN-413 was examined by measuring fluorescein leakage. Results: SZN-413-p induced Wnt/ß-catenin signaling and upregulated blood-brain barrier/blood-retina barrier gene expressions in endothelial cells. In the oxygen-induced retinopathy mouse model, SZN-413-p and SZN-413 significantly reduced the neovascularization area size (P < 0.001) to a level comparable to, or better than the positive control aflibercept. Both agonists also showed a reduction in avascular area size compared to vehicle (P < 0.001) and aflibercept groups (P < 0.05 and P < 0.01 for SZN-413-p and SZN-413, respectively). In the VEGF-induced retinal vascular leakage rabbit model, SZN-413 reduced retinal vascular leakage by ∼80%, compared to the vehicle-treated group (P < 0.01). Conclusions: Reduction of neovascular tufts and avascular areas and of VEGF-driven retinal vascular leakage suggests that SZN-413 can simultaneously address retinal non-perfusion and vascular leakage. Translational Relevance: FZD4 signaling modulation by SZN-413 is a novel mechanism of action that can offer a new therapeutic strategy for diabetic retinopathy.

In Vitro Histone Demethylase Assays.

Dynamic histone methylation regulates gene activation and repression. It is involved in proliferation, differentiation, lineage specification, and development. Histone demethylase assays are invaluable in studying histone demethylation substrate recognition, kinetics, regulation, and inhibition by small molecules, many of which are potential therapeutics. Here we describe general procedures to purify recombinant enzymes from different expression hosts, and to prepare a broad range of substrates, as well as to set up a variety of in vitro histone demethylase assays. These assays provide useful tools for discoveries from enzymes to drugs.

Development of selective bispecific Wnt mimetics for bone loss and repair.

The Wnt signaling pathway is intricately connected with bone mass regulation in humans and rodent models. We designed an antibody-based platform that generates potent and selective Wnt mimetics. Using this platform, we engineer bi-specific Wnt mimetics that target Frizzled and low-density lipoprotein receptor-related proteins and evaluate their effects on bone accrual in murine models. These synthetic Wnt agonists induce rapid and robust bone building effects, and correct bone mass deficiency and bone defects in various disease models, including osteoporosis, aging, and long bone fracture. Furthermore, when these Wnt agonists are combined with antiresorptive bisphosphonates or anti-sclerostin antibody therapies, additional bone accrual/maintenance effects are observed compared to monotherapy, which could benefit individuals with severe and/or acute bone-building deficiencies. Our data support the continued development of Wnt mimetics for the treatment of diseases of low bone mineral density, including osteoporosis.

Context-Dependent Requirement of Euchromatic Histone Methyltransferase Activity during Reprogramming to Pluripotency.

Methylation of histone 3 at lysine 9 (H3K9) constitutes a roadblock for cellular reprogramming. Interference with methyltransferases or activation of demethylases by the cofactor ascorbic acid (AA) facilitates the derivation of induced pluripotent stem cells (iPSCs), but possible interactions between specific methyltransferases and AA treatment remain insufficiently explored. We show that chemical inhibition of the methyltransferases EHMT1 and EHMT2 counteracts iPSC formation in an enhanced reprogramming system in the presence of AA, an effect that is dependent on EHMT1. EHMT inhibition during enhanced reprogramming is associated with rapid loss of H3K9 dimethylation, inefficient downregulation of somatic genes, and failed mesenchymal-to-epithelial transition. Furthermore, transient EHMT inhibition during reprogramming yields iPSCs that fail to efficiently give rise to viable mice upon blastocyst injection. Our observations establish novel functions of H3K9 methyltransferases and suggest that a functional balance between AA-stimulated enzymes and EHMTs supports efficient and less error-prone iPSC reprogramming to pluripotency.

Chromatin Starts to Come Clean.

In an effort to identify a chromatin-associated pluripotent network, Rafiee et al., (2016) developed a powerful ChIP-MS technique and discovered a novel protein, TRIM24, enriched on OCT4-, SOX2-, and NANOG-associated chromatin, paving the way for future proteomic studies on chromatin.

Co-repressor CBFA2T2 regulates pluripotency and germline development.

Developmental specification of germ cells lies at the heart of inheritance, as germ cells contain all of the genetic and epigenetic information transmitted between generations. The critical developmental event distinguishing germline from somatic lineages is the differentiation of primordial germ cells (PGCs), precursors of sex-specific gametes that produce an entire organism upon fertilization. Germ cells toggle between uni- and pluripotent states as they exhibit their own 'latent' form of pluripotency. For example, PGCs express a number of transcription factors in common with embryonic stem (ES) cells, including OCT4 (encoded by Pou5f1), SOX2, NANOG and PRDM14 (refs 2, 3, 4). A biochemical mechanism by which these transcription factors converge on chromatin to produce the dramatic rearrangements underlying ES-cell- and PGC-specific transcriptional programs remains poorly understood. Here we identify a novel co-repressor protein, CBFA2T2, that regulates pluripotency and germline specification in mice. Cbfa2t2(-/-) mice display severe defects in PGC maturation and epigenetic reprogramming. CBFA2T2 forms a biochemical complex with PRDM14, a germline-specific transcription factor. Mechanistically, CBFA2T2 oligomerizes to form a scaffold upon which PRDM14 and OCT4 are stabilized on chromatin. Thus, in contrast to the traditional 'passenger' role of a co-repressor, CBFA2T2 functions synergistically with transcription factors at the crossroads of the fundamental developmental plasticity between uni- and pluripotency.

Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide.

One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homolog of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO.

Single-cell profiling of epigenetic modifiers identifies PRDM14 as an inducer of cell fate in the mammalian embryo.

Cell plasticity or potency is necessary for the formation of multiple cell types. The mechanisms underlying this plasticity are largely unknown. Preimplantation mouse embryos undergo drastic changes in cellular potency, starting with the totipotent zygote through to the formation of the pluripotent inner cell mass (ICM) and differentiated trophectoderm in the blastocyst. Here, we set out to identify and functionally characterize chromatin modifiers that define the transitions of potency and cell fate in the mouse embryo. Using a quantitative microfluidics approach in single cells, we show that developmental transitions are marked by distinctive combinatorial profiles of epigenetic modifiers. Pluripotent cells of the ICM are distinct from their differentiated trophectoderm counterparts. We show that PRDM14 is heterogeneously expressed in 4-cell-stage embryos. Forced expression of PRDM14 at the 2-cell stage leads to increased H3R26me2 and can induce a pluripotent ICM fate. Our results shed light on the epigenetic networks that govern cellular potency and identity in vivo.

Molecular signals of epigenetic states.

Epigenetic signals are responsible for the establishment, maintenance, and reversal of metastable transcriptional states that are fundamental for the cell's ability to "remember" past events, such as changes in the external environment or developmental cues. Complex epigenetic states are orchestrated by several converging and reinforcing signals, including transcription factors, noncoding RNAs, DNA methylation, and histone modifications. Although all of these pathways modulate transcription from chromatin in vivo, the mechanisms by which epigenetic information is transmitted through cell division remain unclear. Because epigenetic states are metastable and change in response to the appropriate signals, a deeper understanding of their molecular framework will allow us to tackle the dysregulation of epigenetics in disease.

Unambiguous determination of isobaric histone modifications by reversed-phase retention time and high-mass accuracy.

Methylation and acetylation of lysines are crucial posttranslational modifications that regulate gene transcription and have been shown to be misregulated in many forms of cancers. Western blot, immunoprecipitation, and immunofluorescence are commonly used to characterize histone acetylation and methylation. However, these approaches are limited by the availability, site specificity, and cross-reactivity of antibodies. Mass spectrometry is emerging as an additional powerful tool for histone characterization. The isobaric nature of trimethylation and acetylation (42.0470 and 42.0106 Da, respectively) confounds histone characterization by means other than high-resolution/high-mass accuracy mass spectrometry. In this study, we adapted methodology that exploits difference in the relative retention time of acetylated and methylated peptides to unequivocally distinguish between these two modifications even with low-mass accuracy mass spectrometers. The approach was tested on tryptic digest of Saccharomyces cerevisiae histones. We found that acetylation resulted in increased retention in reversed-phase chromatography, whereas methylation, including trimethylation, showed little change in retention. For example, the acetylated forms of peptide (27)KSAPSTGGVKKPHR(40) eluted at 15.63 min, whereas the methylated forms eluted at 13.89 min. In addition, the effect of acetylation was cumulative as observed in the case of peptide (9)KSTGGKAPR(17), whose unmodified, monoacetylated, and diacetylated isoforms eluted at 7.43, 10.47, and 16.49 min, respectively. The modification patterns of the peptides in question were subsequently verified by high-mass accuracy tandem mass spectrometry.

The ARID domain of the H3K4 demethylase RBP2 binds to a DNA CCGCCC motif.

The histone H3 lysine 4 demethylase RBP2 contains a DNA binding domain, the AT-rich interaction domain (ARID). We solved the structure of ARID by NMR, identified its DNA binding motif (CCGCCC) and characterized the binding contacts. Immunofluorescence and luciferase assays indicated that ARID is required for RBP2 demethylase activity in cells and that DNA recognition is essential to regulate transcription.

Identification of histone demethylases in Saccharomyces cerevisiae.

Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Delta, gis1Delta, rph1Delta, jhd1Delta, and jhd2Delta (yjr119cDelta)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Delta. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Delta and jhd2Delta strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Delta and jhd1Delta strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.

Unusual four-bond secondary H/D isotope effect supports a short-strong hydrogen bond between phospholipase A2 and a transition state analogue inhibitor.

A prominent secondary four-bond hydrogen/deuterium isotope effect was observed from proton NMR at the active site histidine imidazole ring of bovine pancreatic sPLA(2) in the presence of a phosphonate transition state analogue. The cross-modulation of H(epsilon2)/H48 and H(delta1)/H48 resonances was confirmed by line shape simulation that follows the McConnell equation with fractionation factors incorporated to account for the change in the signal magnitude as well as the resonance line shape at various H(2)O/D(2)O solvent mixtures. While the downfield shift of each individual proton upon deuteration on the opposite site can be attributed to the proton-relay system of the H48-D99 catalytic dyad in sPLA(2), the observation that H(delta1)/H48 induces a 3-fold larger H/D secondary isotope effect ( approximately 0.15 ppm) on H(epsilon2)/H48 than vice versa ( approximately 0.05 ppm) is interpreted as additional spectroscopic evidence for the previously proposed short-strong hydrogen bond formed between the donor N(delta1)/H48 and a nonbridging phosphonate oxygen atom of the transition state analogue. These results provide additional details for the catalytic mechanism of sPLA(2) and demonstrate that the intrinsic H/D secondary isotope effect is a useful tool to probe hydrogen bond strength.