Efficacy and safety of oral insulin versus placebo for patients with diabetes mellitus: A systematic review and meta-analysis.

OBJECTIVE: Compliance to insulin injections is poor due to difficulty in subcutaneous administration. Hence, there is a need of an oral formulation of insulin. Oral insulin is currently under investigation. The present analysis aimed to evaluate oral insulin versus placebo for patients with diabetes mellitus (type-1 and type-2). MATERIALS AND METHODS: Results from PUBMED and MEDLINE were searched and compiled from January 1, 2000 to January 9, 2020. Postprandial blood glucose excursions (2PPG), glycated hemoglobin (HbA1c), C-peptide levels, and immune antibody (IAA) levels were compared between the arms. In addition, time to diabetes and safety of oral insulin were discussed. RESULTS: Thirteen out of 1778 trials were included to the analysis. Oral insulin was found to induce significant reduction in mean 2PPG excursion (standardized mean difference [SMD]: -1.94, 95% CI: -3.20 to -0.68, I2 = 91.81, P < 0.005) and mean IAA levels (SMD:-0.49, 95% CI: -0.82 to -0.16, I2 = 27.12, P < 0.005) compared with placebo. Mean C-peptide levels were notably lower in the oral insulin arm. However, the difference was not statistically significant. No significant difference was observed in mean HbA1c levels. The rate of development of type-1 diabetes was not significantly influenced by oral insulin. No deaths or treatment-related serious adverse events were reported. CONCLUSION: Oral insulin provided significant benefits for acute maintenance of diabetes mellitus. It elicited lower immune response and was well tolerated. This new formulation has potential to augment the management of diabetes mellitus. More studies are required to assess its long-term effects.

Allele-Specific Chemical Rescue of Histone Demethylases Using Abiotic Cofactors.

Closely related protein families evolved from common ancestral genes present a significant hurdle in developing member- and isoform-specific chemical probes, owing to their similarity in fold and function. In this piece of work, we explore an allele-specific chemical rescue strategy to activate a "dead" variant of a wildtype protein using synthetic cofactors and demonstrate its successful application to the members of the alpha-ketoglutarate (αKG)-dependent histone demethylase 4 (KDM4) family. We show that a mutation at a specific residue in the catalytic site renders the variant inactive toward the natural cosubstrate. In contrast, αKG derivatives bearing appropriate stereoelectronic features endowed the mutant with native-like demethylase activity while remaining refractory to a set of wild type dioxygenases. The orthogonal enzyme-cofactor pairs demonstrated site- and degree-specific lysine demethylation on a full-length chromosomal histone in the cellular milieu. Our work offers a strategy to modulate a specific histone demethylase by identifying and engineering a conserved phenylalanine residue, which acts as a gatekeeper in the KDM4 subfamily, to sensitize the enzyme toward a novel set of αKG derivatives. The orthogonal pairs developed herein will serve as probes to study the role of degree-specific lysine demethylation in mammalian gene expression. Furthermore, this approach to overcome active site degeneracy is expected to have general application among all human αKG-dependent dioxygenases.

Catalytic Space Engineering as a Strategy to Activate C-H Oxidation on 5-Methylcytosine in Mammalian Genome.

Conditional remodeling of enzyme catalysis is a formidable challenge in protein engineering. Herein, we have undertaken a unique active site engineering tactic to command catalytic outcomes. With ten-eleven translocation (TET) enzyme as a paradigm, we show that variants with an expanded active site significantly enhance multistep C-H oxidation in 5-methylcytosine (5mC), whereas a crowded cavity leads to a single-step catalytic apparatus. We further identify an evolutionarily conserved residue in the TET family with a remarkable catalysis-directing ability. The activating variant demonstrated its prowess to oxidize 5mC in chromosomal DNA for potentiating expression of genes including tumor suppressors.

Engineering bromodomains with a photoactive amino acid by engaging 'Privileged' tRNA synthetases.

Site-specific placement of unnatural amino acids, particularly those responsive to light, offers an elegant approach to control protein function and capture their fleeting 'interactome'. Herein, we have resurrected 4-(trifluoromethyldiazirinyl)-phenylalanine, an underutilized photo-crosslinker, by introducing several key features including easy synthetic access, site-specific incorporation by 'privileged' synthetases and superior crosslinking efficiency, to develop photo-crosslinkable bromodomains suitable for 'interactome' profiling.

Synthesis of 5-Dihydroxyboryluridine Phosphoramidite and Its Site-Specific Incorporation into Oligonucleotides for Probing Thymine DNA Glycosylase.

A concise synthetic strategy to 5-dihydroxyboryldexoyuridine (5boU) phosphoramidite has been developed. 5boU was introduced into short oligonucleotides in a site-specific manner, demonstrating compatibility of the boronic acid moiety with standard solid-phase DNA synthesis chemistry. Electrophilic 5boU DNAs inhibited thymine DNA glycosylase, a cancer-relevant DNA-modifying enzyme. We envisage diverse applications of 5boU in organic synthesis, medicinal chemistry, and chemical biology.

Site- and degree-specific C-H oxidation on 5-methylcytosine homologues for probing active DNA demethylation.

Ten-eleven translocation (TET) enzymes oxidize C-H bonds in 5-methylcytosine (5mC) to hydroxyl (5hmC), formyl (5fC) and carboxyl (5caC) intermediates en route to DNA demethylation. It has remained a challenge to study the function of a single oxidized product. We investigate whether alkyl groups other than methyl could be oxidized by TET proteins to generate a specific intermediate. We report here that TET2 oxidizes 5-ethylcytosine (5eC) only to 5-hydroxyethylcytosine (5heC). In biochemical assays, 5heC acts as a docking site for proteins implicated in transcription, imbuing this modification with potential gene regulatory activity. We observe that 5heC is resistant to downstream wild type hydrolases, but not to the engineered enzymes, thus establishing a unique tool to conditionally alter the stability of 5heC on DNA. Furthermore, we devised a chemical approach for orthogonal labeling of 5heC. Our work offers a platform for synthesis of novel 5-alkylcytosines, provides an approach to 'tame' TET activity, and identifies 5heC as an unnatural modification with a potential to control chromatin-dependent processes.

Site-specific azide-acetyllysine photochemistry on epigenetic readers for interactome profiling.

Chemical modifications on DNA, RNA and histones are recognized by an array of 'reader' modules to regulate transcriptional programming and cell fate. However, identification of reader-specific interacting partners in a dynamic cellular environment remains a significant challenge. Herein, we report a chemoproteomic approach termed 'interaction-based protein profiling' (IBPP) to characterize novel interacting partners of potentially any reader protein. IBPP harnesses a photosensitive amino acid introduced into the hydrophobic pocket of a reader module to crosslink and enrich transient interacting partners that are inaccessible to traditional methods. Using bromodomain-containing protein 4 (BRD4) as a paradigm, we engineer an 'aromatic cage' of the bromodomain to introduce 4-azido-l-phenylalanine (pAzF) without compromising its ability to recognize acetylated lysine residues in histone proteins. We establish the binding efficiency, substrate specificity and crosslinking ability of the engineered 'reader' module in biochemical assays. Applying IBPP, we uncovered novel acetylated interacting partners of BRD4, such as transcription factors, expanding on its previously unappreciated role in diverse biological processes. By setting up an azide-acetyllysine photoreaction deep inside the bromodomain aromatic cage as a means to detect protein acetylation, our approach provides a potentially general platform for rapid and unbiased profiling of interacting partners of diverse epigenetic readers whose functions in eukaryotic gene regulation remain convoluted.

A rapid mass spectrometric method for the measurement of catalytic activity of ten-eleven translocation enzymes.

Enzymatic methylation at carbon five on cytosine (5mC) in DNA is a hallmark of mammalian epigenetic programming and is critical to gene regulation during early embryonic development. It has recently been shown that dynamic erasure of 5mC by three members of the ten-eleven translocation (TET) family plays a key role in cellular differentiation. TET enzymes belong to Fe (II)- and 2-ketoglutarate (2KG) dependent dioxygenases that successively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5CaC), thus providing a chemical basis for the removal of 5mC which once was thought to be a permanent mark in mammalian genome. Since then a wide range of biochemical assays have been developed to characterize TET activity. Majority of these methods require multi-step processing to detect and quantify the TET-mediated oxidized products. In this study, we have developed a MALDI mass spectrometry based method that directly measures the TET activity with high sensitivity while eliminating the need for any intermediate processing steps. We applied this method to the measurement of enzymatic activity of TET2 and 3, Michaleis-Menten parameters (KM and kcat) of TET-2KG pairs and inhibitory concentration (IC50) of known small-molecule inhibitors of TETs. We further demonstrated the suitability of the assay to analyze chemoenzymatic labeling of 5hmC by ß-glucosyltransferase, highlighting the potential for broad application of our method in deconvoluting the functions of novel DNA demethylases.

Engineering Biological C-H Functionalization Leads to Allele-Specific Regulation of Histone Demethylases.

Oxidative C-H hydroxylation of methyl groups, followed by their removal from DNA, RNA, or histones, is an epigenetic process critical to transcriptional reprogramming and cell fate determination. This reaction is catalyzed by Fe(II)-dependent dioxygenases using the essential metabolite 2-ketoglutarate (2KG) as a cofactor. Given that the human genome encodes for more than 60 2KG-dependent dioxygenases, assigning their individual functions remains a significant challenge. Here we describe a protein-ligand interface engineering approach to break the biochemical degeneracy of these enzymes. Using histone lysine demethylase 4 as a proof-of-concept, we show that the enzyme active site can be expanded to employ bulky 2KG analogues that do not sensitize wild-type demethylases. We establish the orthogonality, substrate specificity, and catalytic competency of the engineered demethylation apparatus in biochemical assays. We further demonstrate demethylation of cognate substrates in physiologically relevant settings. Our results provide a paradigm for rapid and conditional manipulation of histone demethylases to uncloak their isoform-specific functions.

α-Trimethylsilylmethylamine radical cation in the synthesis of cyclic amines and beyond.

The evolution of chemistry associated with the photoinduced electron transfer (PET)-generated α-trimethylsilylmethylamine radical cation cyclization to a tethered olefin to synthesize cyclic amine structural frame works is presented in chronological order. The importance of this interesting chemistry is demonstrated by the synthesis of several novel glycosidase inhibitors.