Chemical Methods to Identify Epigenetic Modifications in Cytosine Bases.

Chemical modifications to Cytosine bases are among the most studied epigenetic markers and their detection in the human genome plays a crucial role in gaining more insights about gene regulation, prognosis of genetic disorders and unraveling genetic inheritance patterns. The Cytosine methylated at the 5th position and oxidized derivatives thereof generated in the demethylation pathways, perform separate and unique epigenetic functions in an organism. As the presence of various Cytosine modifications is associated with diverse diseases, including cancer, there has been a strong focus on developing methods, both chemical and alternative approaches, capable of detecting these modifications at a single-base resolution across the entire genome. In this comprehensive review, we aim to consolidate the various chemical methods and understanding their chemistry that have been established to date for the detection of various Cytosine modifications.

Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses.

Pancreatic α-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human α-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality α-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live α-cells from dissociated human islet cells with ~95% purity. The α-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form α-pseudoislets. The α-pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key α-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in α-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary α-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.

A Chemical Model of a TET Enzyme for Selective Oxidation of Hydroxymethyl Cytosine to Formyl Cytosine.

Methylation/demethylation of cytosines in DNA is central to epigenetics, which plays crucial roles in the regulation of about half of all human genes. Although the methylation mechanism, which downregulates gene expression, has been sufficiently decoded; the demethylation pathway, which upregulates gene expression, still holds questions to be answered. Demethylation of 5-methylcytosine by ten-eleven translocation (TET) enzymes yields understudied but epigenetically relevant intermediates, 5-hydroxymethyl (5-hmC), 5-formyl (5-fC), and 5-carboxyl (5-caC) cytosines. Here we report an iron complex, FeIIITAML (TAML = tetraamido macrocyclic ligand), which can facilitate selective oxidation of 5-hmC to its oxidative derivatives by forming a high-valent Fe-oxo intermediate in the presence of H2O2 under physiologically relevant conditions. Detailed HPLC analyses supported by a wide reaction condition optimization for the 5-hmC → 5-fC oxidation provides us with a chemical model of the TET enzyme. This study shines light on future efforts for a better understanding of the roles of 5-hmC and the TET enzyme mechanism and potentially novel therapeutic methods.

Optochemical control of Cu(I) homeostasis in mammalian cells.

Copper can act as a double-edged sword as it can cause fatal diseases when in excess or shortage. Precise control of copper homeostasis is maintained by a complex machinery inside cells. To overcome imbalances in copper concentration, we have developed a simple system to control the cellular copper concentration by using a photocaged chelator and light. This photocaged chelator allowed us to control cellular copper concentration in a spatiotemporal manner.

Detailed Insights into the Inhibitory Mechanism of New Ebselen Derivatives against Main Protease (Mpro) of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2).

SARS-CoV-2 main protease (Mpro/3CLpro) is a crucial target for therapeutics, which is responsible for viral polyprotein cleavage and plays a vital role in virus replication and survival. Recent studies suggest that 2-phenylbenzisoselenazol-3(2H)-one (ebselen) is a potent covalent inhibitor of Mpro, which affects its enzymatic activity and virus survival. Herein, we synthesized various ebselen derivatives to understand the mechanism of Mpro inhibition by ebselen. Using ebselen derivatives, we characterized the detailed interaction mechanism with Mpro. We discovered that modification of the parent ebselen inhibitor with an electron-withdrawing group (NO2) increases the inhibition efficacy by 2-fold. We also solved the structure of an Mpro complex with an ebselen derivative showing the mechanism of inhibition by blocking the catalytic Cys145 of Mpro. Using a combination of crystal structures and LC-MS data, we showed that Mpro hydrolyzes the new ebselen derivative and leaves behind selenium (Se) bound with Cys145 of the catalytic dyad of Mpro. We also described the binding profile of ebselen-based inhibitors using molecular modeling predictions supported by binding and inhibition assays. Furthermore, we have also solved the crystal structure of catalytically inactive mutant H41N-Mpro, which represents the inactive state of the protein where the substrate binding pocket is blocked. The inhibited structure of H41N-Mpro shows gatekeeper residues in the substrate binding pocket responsible for blocking the substrate binding; mutation of these gatekeeper residues leads to hyperactive Mpro.

Targeted protein degradation using the lysosomal pathway.

Degradation strategies have shown enormous promise after the inception of molecules like PROTACs (PRoteolysis TArgeting Chimeras) that induce the degradation of the substrate of choice rather than depending on blocking their catalytic activity like conventional inhibitory drugs. Over the past two decades, the application of PROTACs has made quite an impact, even reaching clinical translations. However, a major class of macromolecular targets, be that large proteins, aggregates, organelles or non-protein substrates, remain untouched when utilizing the ubiquitin-proteasomal pathway of degradation. In this review, we have attempted to cover modalities of targeted degradation that instead focus on recruiting the lysosomal pathway of degradation, which is gaining importance and being explored extensively as alternate and efficient approaches for treating disease-related milieus.

Repurposing pinacol esters of boronic acids for tuning viscoelastic properties of glucose-responsive polymer hydrogels: effects on insulin release kinetics.

In the era of the diabetes pandemic, injectable hydrogels (HGs) capable of releasing the desired amount of insulin under hyperglycemic conditions will significantly advance smart insulin development. Several smart boronic acid-based polymer HGs release insulin under high-glucose conditions. However, the correlation of insulin release characteristics with rheological properties is not well understood yet. Herein, we report a generalized and facile fabrication strategy of a new class of glucose-responsive hydrogels by crosslinking a biocompatible polymer, poly(vinyl alcohol) with pinacol esters of bisboronic acids via transesterification reactions. We show the versatility of the method by fabricating four hydrogels with diverse rheological properties. The HGs embody more than 70% water amenable for hosting insulin in the matrix. HG with high storage modulus, derived from 1,4-benzenediboronic acid bis(pinacol) ester releases ∼3 fold less insulin compared to softer HGs derived from acetylene-1,2-diyl bis(boronic acid pinacol ester) and bis[(pinacolato)boryl]methane under hyperglycemic conditions. We find that HG derived from the bis[(pinacolato)boryl]methane crosslinker exhibits superior insulin release properties due to the softness of the hydrogel matrix. We further show that the newly formulated gel is injectable without any structural change in the released insulin molecules and does not cause cytotoxicity. We believe that glucose-responsive hydrogels with tunable viscoelastic properties will pave the way for developing a variety of hydrogels with programmable insulin release properties.

LYTACs: An Emerging Tool for the Degradation of Non-Cytosolic Proteins.

In the last two decades, targeted protein degradation has rapidly gained popularity as a technique to eliminate disease-causing undruggable proteins. Over the years, many tools have been devised to degrade proteins by exploiting natural protein homeostasis machinery available in our body, with LYTACs being the latest to come on board. LYTACs, or lysosome-targeting chimeras, make use of the lysosome degradation pathway by recruiting proteins to lysosome-shuttling receptors located at the cell surface. LYTACs are specifically meant for the degradation of membrane-bound and extracellular proteins, which account for the products of 40 % of all protein-encoding genes. In this highlight, we describe two studies that demonstrate the scope of LYTACs and its advantages over the other protein degradation platforms. In the first study, the LYTAC utilizes the cation-independent mannose-6-phosphate receptor (CI-M6PR), while the second study uses the asialoglycoprotein receptor (ASGPR) which is found only on the surface of liver cells.

Harnessing reaction-based probes to preferentially target pancreatic ß-cells and ß-like cells.

Highly sensitive approaches to target insulin-expressing cells would allow more effective imaging, sorting, and analysis of pancreatic ß-cells. Here, we introduce the use of a reaction-based probe, diacetylated Zinpyr1 (DA-ZP1), to image pancreatic ß-cells and ß-like cells derived from human pluripotent stem cells. We harness the high intracellular zinc concentration of ß-cells to induce a fluorescence signal in cells after administration of DA-ZP1. Given its specificity and rapid uptake by cells, we used DA-ZP1 to purify live stem cell-derived ß-like cells as confirmed by immunostaining analysis. We tested the ability of DA-ZP1 to image transplanted human islet grafts and endogenous mouse pancreatic islets in vivo after its systemic administration into mice. Thus, DA-ZP1 enables purification of insulin-secreting ß-like cells for downstream applications, such as functional studies, gene-expression, and cell-cell interaction analyses and can be used to label engrafted human islets and endogenous mouse islets in vivo.

Controlling PROTACs with Light.

Proteolysis targeting chimeras, PROTACs, are emerging as a powerful strategy for exerting exogenous control over protein levels, allowing small molecules to exploit the ubiquitin-proteasome pathway for targeted protein degradation. This highlight focuses on the fusion of photochemistry with these bifunctional compounds, which has provided a novel pathway for spatiotemporally tuning the activation of PROTACs in the form of their photocaged and photoswitchable versions. Photocaged PROTACs consist of a hindered optolabile group that detaches only upon irradiation at a specific wavelength, releasing the active PROTAC. These modified PROTACs are inactive in the dark. Photoswitchable PROTACs are photoisomerizable molecules with azobenzene linkages that are active in either the cis or trans form and inactive in the other. The isomers interconvert upon irradiation with an appropriate wavelength of light and relax to the thermodynamically stable isomer in the dark or with another wavelength of light. Although photocaged PROTACs only permit activation control for protein degradation, photoswitching PROTACs offer reversible activation and deactivation by using suitable wavelengths of light.

Optochemical Control of Protein Degradation.

Optochemical approach has been successfully utilized to regulate cellular protein degradation with a high resolution of spatiotemporal control. In this highlight, we discuss two recent developments of combining reversible optochemical functionalities with the bifunctional proteolysis targeting chimeras, or PROTACs to achieve light-controlled degradation of protein targets of interest. PHOTACs are azobeneze-containing molecules that are inactive as trans forms and active as cis forms, switchable upon pulse-irradiation with either an activating 390 nm light or a deactivating 525 nm light. In contrast, photoPROTACs are o-F4 -azobenzene-containing molecules that can be switched between active trans isomers and inactive cis isomers by a single irradiation event using 415 or 530 nm light. Combining these two optochemically controlled PROTAC systems has the potential to achieve orthogonal control on protein degradation.

Native Zinc Catalyzes Selective and Traceless Release of Small Molecules in ß-Cells.

The loss of insulin-producing ß-cells is the central pathological event in type 1 and 2 diabetes, which has led to efforts to identify molecules to promote ß-cell proliferation, protection, and imaging. However, the lack of ß-cell specificity of these molecules jeopardizes their therapeutic potential. A general platform for selective release of small-molecule cargoes in ß-cells over other islet cells ex vivo or other cell-types in an organismal context will be immensely valuable in advancing diabetes research and therapeutic development. Here, we leverage the unusually high Zn(II) concentration in ß-cells to develop a Zn(II)-based prodrug system to selectively and tracelessly deliver bioactive small molecules and fluorophores to ß-cells. The Zn(II)-targeting mechanism enriches the inactive cargo in ß-cells as compared to other pancreatic cells; importantly, Zn(II)-mediated hydrolysis triggers cargo activation. This prodrug system, with modular components that allow for fine-tuning selectivity, should enable the safer and more effective targeting of ß-cells.

Halogen Bonding in Biomimetic Deiodination of Thyroid Hormones and their Metabolites and Dehalogenation of Halogenated Nucleosides.

Thyroid hormones (THs) are key players in the endocrine system and play pivotal roles in carbohydrate and fat metabolism, protein synthesis, overall growth, and brain development. The thyroid gland predominantly produces thyroxine or 3,5,3',5'-tetraiodothyronine (T4) as a prohormone; three isoforms of a mammalian selenoenzyme-iodothyronine deiodinase (DIO1, DIO2 and DIO3)-catalyze the regioselective deiodination of T4 to produce biologically active and inactive metabolites. Whereas DIO1 catalyzes both 5- and 5'-deiodination of T4, DIO2 and DIO3 selectively mediate 5- and 5'-deiodination, respectively. In this review we discuss the regioselective deiodination of THs in the presence of organochalcogen compounds. Naphthalene-based compounds containing sulfur and/or selenium at the peri positions mediate regioselective 5-deiodination of THs, detailed mechanistic studies having revealed that the heterolytic cleavage of the C-I bond is facilitated by the formation of cooperative Se/S⋅⋅⋅I halogen bonds and Se/S⋅⋅⋅Se chalcogen bonds. We also discuss the biomimetic deiodination of several TH metabolites, including sulfated THs, iodothyronamines, and iodotyrosines. A brief discussion on the dehalogenation of halogenated nucleosides and nucleobases in the presence of organochalcogen compounds is also included.

A Singular System with Precise Dosing and Spatiotemporal Control of CRISPR-Cas9.

Several genome engineering applications of CRISPR-Cas9, an RNA-guided DNA endonuclease, require precision control of Cas9 activity over dosage, timing, and targeted site in an organism. While some control of Cas9 activity over dose and time have been achieved using small molecules, and spatial control using light, no singular system with control over all the three attributes exists. Furthermore, the reported small-molecule systems lack wide dynamic range, have background activity in the absence of the small-molecule controller, and are not biologically inert, while the optogenetic systems require prolonged exposure to high-intensity light. We previously reported a small-molecule-controlled Cas9 system with some dosage and temporal control. By photocaging this Cas9 activator to render it biologically inert and photoactivatable, and employing next-generation protein engineering approaches, we have built a system with a wide dynamic range, low background, and fast photoactivation using a low-intensity light while rendering the small-molecule activator biologically inert. We anticipate these precision controls will propel the development of practical applications of Cas9.

Precision Control of CRISPR-Cas9 Using Small Molecules and Light.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas system is an adaptive immune system of bacteria that has furnished several RNA-guided DNA endonucleases (e.g., Cas9) that are revolutionizing the field of genome engineering. Cas9 is being used to effect genomic alterations as well as in gene drives, where a particular trait may be propagated through a targeted species population over several generations. The ease of targeting catalytically impaired Cas9 to any genomic loci has led to development of technologies for base editing, chromatin imaging and modeling, epigenetic editing, and gene regulation. Unsurprisingly, Cas9 is being developed for numerous applications in biotechnology and biomedical research and as a gene therapy agent for multiple pathologies. There is a need for precise control of Cas9 activity over several dimensions, including those of dose, time, and space in these applications. Such precision controls, which are required of therapeutic agents, are particularly important for Cas9 as off-target effects, chromosomal translocations, immunogenic response, genotoxicity, and embryonic mosaicism are observed at elevated levels and with prolonged activity of Cas9. Here, we provide a perspective on advances in the precision control of Cas9 over aforementioned dimensions using external stimuli (e.g., small molecules or light) for controlled activation, inhibition, or degradation of Cas9.

Reversible trapping and reaction acceleration within dynamically self-assembling nanoflasks.

The chemical behaviour of molecules can be significantly modified by confinement to volumes comparable to the dimensions of the molecules. Although such confined spaces can be found in various nanostructured materials, such as zeolites, nanoporous organic frameworks and colloidal nanocrystal assemblies, the slow diffusion of molecules in and out of these materials has greatly hampered studying the effect of confinement on their physicochemical properties. Here, we show that this diffusion limitation can be overcome by reversibly creating and destroying confined environments by means of ultraviolet and visible light irradiation. We use colloidal nanocrystals functionalized with light-responsive ligands that readily self-assemble and trap various molecules from the surrounding bulk solution. Once trapped, these molecules can undergo chemical reactions with increased rates and with stereoselectivities significantly different from those in bulk solution. Illumination with visible light disassembles these nanoflasks, releasing the product in solution and thereby establishes a catalytic cycle. These dynamic nanoflasks can be useful for studying chemical reactivities in confined environments and for synthesizing molecules that are otherwise hard to achieve in bulk solution.

Light-controlled self-assembly of non-photoresponsive nanoparticles.

The ability to guide the assembly of nanosized objects reversibly with external stimuli, in particular light, is of fundamental importance, and it contributes to the development of applications as diverse as nanofabrication and controlled drug delivery. However, all the systems described to date are based on nanoparticles (NPs) that are inherently photoresponsive, which makes their preparation cumbersome and can markedly hamper their performance. Here we describe a conceptually new methodology to assemble NPs reversibly using light that does not require the particles to be functionalized with light-responsive ligands. Our strategy is based on the use of a photoswitchable medium that responds to light in such a way that it modulates the interparticle interactions. NP assembly proceeds quantitatively and without apparent fatigue, both in solution and in gels. Exposing the gels to light in a spatially controlled manner allowed us to draw images that spontaneously disappeared after a specific period of time.

Selenium-Mediated Dehalogenation of Halogenated Nucleosides and its Relevance to the DNA Repair Pathway.

Halogenated nucleosides can be incorporated into the newly synthesized DNA of replicating cells and therefore are commonly used in the detection of proliferating cells in living tissues. Dehalogenation of these modified nucleosides is one of the key pathways involved in DNA repair mediated by the uracil-DNA glycosylase. Herein, we report the first example of a selenium-mediated dehalogenation of halogenated nucleosides. We also show that the mechanism for the debromination is remarkably different from that of deiodination and that the presence of a ribose or deoxyribose moiety in the nucleosides facilitates the deiodination. The results described herein should help in understanding the metabolism of halogenated nucleosides in DNA and RNA.

Orthogonal light-induced self-assembly of nanoparticles using differently substituted azobenzenes.

Precise control of the self-assembly of selected components within complex mixtures is a challenging goal whose realization is important for fabricating novel nanomaterials. Herein we show that by decorating the surfaces of metallic nanoparticles with differently substituted azobenzenes, it is possible to modulate the wavelength of light at which the self-assembly of these nanoparticles is induced. Exposing a mixture of two types of nanoparticles, each functionalized with a different azobenzene, to UV or blue light induces the selective self-assembly of only one type of nanoparticles. Irradiation with the other wavelength triggers the disassembly of the aggregates, and the simultaneous self-assembly of nanoparticles of the other type. By placing both types of azobenzenes on the same nanoparticles, we created unique materials ("frustrated" nanoparticles) whose self-assembly is induced irrespective of the wavelength of the incident light.

Halogen bonding controls the regioselectivity of the deiodination of thyroid hormones and their sulfate analogues.

The type 1 iodothyronine deiodinase (1D-1) in liver and kidney converts the L-thyroxine (T4), a prohormone, by outer-ring (5') deiodination to biologically active 3,3',5-triiodothyronine (T3) or by inner-ring (5) deiodination to inactive 3,3',5'-triiodothronine (rT3). Sulfate conjugation is an important step in the irreversible inactivation of thyroid hormones. While sulfate conjugation of the phenolic hydroxyl group stimulates the 5-deiodination of T4 and T3, it blocks the 5'-deiodination of T4. We show that thyroxine sulfate (T4S) undergoes faster deiodination as compared to the parent thyroid hormone T4 by synthetic selenium compounds. It is also shown that ID-3 mimics, which are remarkably selective to the inner-ring deiodination of T4 and T3, changes the selectivity completely when T4S is used as a substrate. From the theoretical investigations, it is observed that the strength of halogen bonding increases upon sulfate conjugation, which leads to a change in the regioselectivity of ID-3 mimics towards the deiodination of T4S. It has been shown that these mimics perform both the 5'- and 5-ring deiodinations by an identical mechanism.