Overcoming oral insulin delivery barriers: application of cell penetrating peptide and silica-based nanoporous composites

Oral insulin delivery has received the most attention in insulin formulations due to its high patient compliance and, more importantly, to its potential to mimic the physiologic insulin secretion seen in non-diabetic individuals. However, oral insulin delivery has two major limitations: the enzymatic barrier that leads to rapid insulin degradation, and the mucosal barrier that limits insulin’s bioavailability. Several approaches have been actively pursued to circumvent the enzyme barrier, with some of them receiving promising results. Yet, thus far there has been no major success in overcoming the mucosal barrier, which is the main cause in undercutting insulin’s oral bioavailability. In this review of our group’s research, an innovative silica-based, mucoadhesive oral insulin formulation with encapsulated-insulin/cell penetrating peptide (CPP) to overcome both enzyme and mucosal barriers is discussed, and the preliminary and convincing results to confirm the plausibility of this oral insulin delivery system are reviewed. In vitro studies demonstrated that the CPP-insulin conjugates could facilitate cellular uptake of insulin while keeping insulin’s biologic functions intact. It was also confirmed that low molecular weight protamine (LMWP) behaves like a CPP peptide, with a cell translocation potency equivalent to that of the widely studied TAT. The mucoadhesive properties of the produced silica-chitosan composites could be controlled by varying both the pH and composition; the composite consisting of chitosan (25 wt-%) and silica (75 wt-%) exhibited the greatest mucoadhesion at gastric pH. Furthermore, drug release from the composite network could also be regulated by altering the chitosan content. Overall, the universal applicability of those technologies could lead to development of a generic platform for oral delivery of many other bioactive compounds, especially for peptide or protein drugs which inevitably encounter the poor bioavailability issues.     

Signaling effects of demethylasterriquinone B1, a selective insulin receptor modulator

A possible breakthrough in the treatment of diabetes was made with the discovery that a fungal natural product, demethylasterriquinone B1 (DAQ B1), is an orally active, small-molecule mimic of insulin. Subsequent work has shown that the glucose-lowering effects of DAQ B1 are not accompanied by enhanced vascular proliferation, which is a side effect of chronic insulin administration that can lead to arteriosclerosis. Our recent short and modular total synthesis of DAQ B1 could be readily modified to create congeners and afforded ample supplies of the natural product, which permitted intracellular signal transduction of DAQ B1 to be examined. The activities of DAQ B1 and over a dozen related structures were studied for insulin receptor (IR) and insulin receptor substrate-1 phosphorylation. Examination of the effect of DAQ B1 on kinases downstream of the IR in insulin signal transduction showed selective activation of Akt kinase (a metabolic effect) but not of extracellular-regulated kinase (a proliferative effect). The influence of DAQ B1 on gene expression (determined by a microarray study) was also divergent from that of insulin, which activates both proliferative and metabolic pathways. The action of DAQ B1 as a selective insulin receptor modulator can be accounted for by its ability to selectively activate one kinase among the many emanating from insulin receptor autophosphorylation and its reduced effects on gene expression.     

Nature Inspired Small Molecules for Chemical Biology

Chemical biology and drug discovery are instrumental sciences to address unmet medical needs and to gain a deeper understanding of normal and disease state biology in mammalian systems. Unlike most genetic tools, the small molecule modulation of biology is reversible, controllable in space, time and quantity, avoids the removal of gene products from cellular systems and thus enables perturbation of biology in its native state. Natural products, their derivatives as well as small molecules based on the core‐scaffolds of natural products including natural product fragments allow targeting unique, biologically relevant fractions of chemical space that may deliver quality tool compounds. In this essay, we discuss various synthesis approaches inspired by natural products to deliver biologically active small molecules. We argue and provide evidence that inspiration by natural product structure remains a powerful guiding principle for the development of novel approaches to the study biology by means of novel bioactive small molecules.     

Chemical Translational Biology: Redefining Druggability of Protein-Protein Interactions

Chemical biologists use chemical tools to answer biological questions. The translational application of these principles has led to an explosion in the discovery and druggability of new protein targets, including protein-protein interactions (PPIs). Proteins tend to interact with other macromolecules using relatively large and featureless binding surfaces, which has hampered traditional drug discovery efforts, particularly for interactions with weaker affinity. In this article, I discuss several emerging strategies for targeting PPIs, including computational and structural methods and novel screening approaches. In particular, I focus on hijacking intrinsic protein allosteric pathways for the discovery and design of small-molecule and peptide ligands.     

Insulin Signaling as a Therapeutic Target in Glaucomatous Neurodegeneration

Glaucoma is a multifactorial disease that is conventionally managed with treatments to lower intraocular pressure (IOP). Despite these efforts, many patients continue to lose their vision. The degeneration of retinal ganglion cells (RGCs) and their axons in the optic tract that characterizes glaucoma is similar to neurodegeneration in other age-related disorders of the central nervous system (CNS). Identifying the different molecular signaling pathways that contribute to early neuronal dysfunction can be utilized for neuroprotective strategies that prevent degeneration. The discovery of insulin and its receptor in the CNS and retina led to exploration of the role of insulin signaling in the CNS. Historically, insulin was considered a peripherally secreted hormone that regulated glucose homeostasis, with no obvious roles in the CNS. However, a growing number of pre-clinical and clinical studies have demonstrated the potential of modulating insulin signaling in the treatment of neurodegenerative diseases. This review will highlight the role that insulin signaling plays in RGC neurodegeneration. We will focus on how this pathway can be therapeutically targeted to promote RGC axon survival and preserve vision.     

The Biochemistry of Phytocannabinoids and Metabolic Engineering of Their Production in Heterologous Systems

The medicinal properties of cannabis and the its legal status in several countries and jurisdictions has spurred the massive growth of the cannabis economy around the globe. The value of cannabis stems from its euphoric activity offered by the unique phytocannabinoid tetrahydrocannabinol (THC). However, this is rapidly expanding beyond THC owing to other non-psychoactive phytocannabinoids with new bioactivities that will contribute to their development into clinically useful drugs. The discovery of the biosynthesis of major phytocannabinoids has allowed the exploration of their heterologous production by synthetic biology, which may lead to the industrial production of rare phytocannabinoids or novel synthetic cannabinoid pharmaceuticals that are not easily offered by cannabis plants. This review summarizes the biosynthesis of major phytocannabinoids in detail, the most recent development of their metabolic engineering in various systems, and the engineering approaches and strategies used to increase the yield.     

Insulin Induces Phosphorylation of Serine Residues of Translationally Controlled Tumor Protein in 293T Cells

Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP) inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser) residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP) was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation.     

Strategies to Tackle Antimicrobial Resistance: The Example of Escherichia coli and Pseudomonas aeruginosa

Traditional antimicrobial treatments consist of drugs which target different essential functions in pathogens. Nevertheless, bacteria continue to evolve new mechanisms to evade this drug-mediated killing with surprising speed on the deployment of each new drug and antibiotic worldwide, a phenomenon called antimicrobial resistance (AMR). Nowadays, AMR represents a critical health threat, for which new medical interventions are urgently needed. By 2050, it is estimated that the leading cause of death will be through untreatable AMR pathogens. Although antibiotics remain a first-line treatment, non-antibiotic therapies such as prophylactic vaccines and therapeutic monoclonal antibodies (mAbs) are increasingly interesting alternatives to limit the spread of such antibiotic resistant microorganisms. For the discovery of new vaccines and mAbs, the search for effective antigens that are able to raise protective immune responses is a challenging undertaking. In this context, outer membrane vesicles (OMV) represent a promising approach, as they recapitulate the complete antigen repertoire that occurs on the surface of Gram-negative bacteria. In this review, we present Escherichia coli and Pseudomonas aeruginosa as specific examples of key AMR threats caused by Gram-negative bacteria and we discuss the current status of mAbs and vaccine approaches under development as well as how knowledge on OMV could benefit antigen discovery strategies.     

Targeting Insulin-Degrading Enzyme in Insulin Clearance

Hepatic insulin clearance, a physiological process that in response to nutritional cues clears ~50–80% of circulating insulin, is emerging as an important factor in our understanding of the pathogenesis of type 2 diabetes mellitus (T2DM). Insulin-degrading enzyme (IDE) is a highly conserved Zn²⁺-metalloprotease that degrades insulin and several other intermediate-size peptides. Both, insulin clearance and IDE activity are reduced in diabetic patients, albeit the cause-effect relationship in humans remains unproven. Because historically IDE has been proposed as the main enzyme involved in insulin degradation, efforts in the development of IDE inhibitors as therapeutics in diabetic patients has attracted attention during the last decades. In this review, we retrace the path from Mirsky’s seminal discovery of IDE to the present, highlighting the pros and cons of the development of IDE inhibitors as a pharmacological approach to treating diabetic patients.     

Genomics and Epigenomics of Gestational Diabetes Mellitus: Understanding the Molecular Pathways of the Disease Pathogenesis

One of the most common complications during pregnancy is gestational diabetes mellitus (GDM), hyperglycemia that occurs for the first time during pregnancy. The condition is multifactorial, caused by an interaction between genetic, epigenetic, and environmental factors. However, the underlying mechanisms responsible for its pathogenesis remain elusive. Moreover, in contrast to several common metabolic disorders, molecular research in GDM is lagging. It is important to recognize that GDM is still commonly diagnosed during the second trimester of pregnancy using the oral glucose tolerance test (OGGT), at a time when both a fetal and maternal pathophysiology is already present, demonstrating the increased blood glucose levels associated with exacerbated insulin resistance. Therefore, early detection of metabolic changes and associated epigenetic and genetic factors that can lead to an improved prediction of adverse pregnancy outcomes and future cardio-metabolic pathologies in GDM women and their children is imperative. Several genomic and epigenetic approaches have been used to identify the genes, genetic variants, metabolic pathways, and epigenetic modifications involved in GDM to determine its etiology. In this article, we explore these factors as well as how their functional effects may contribute to immediate and future pathologies in women with GDM and their offspring from birth to adulthood. We also discuss how these approaches contribute to the changes in different molecular pathways that contribute to the GDM pathogenesis, with a special focus on the development of insulin resistance.     

Insulin Signal Transduction Perturbations in Insulin Resistance

Type 2 diabetes mellitus is a widespread medical condition, characterized by high blood glucose and inadequate insulin action, which leads to insulin resistance. Insulin resistance in insulin-responsive tissues precedes the onset of pancreatic β-cell dysfunction. Multiple molecular and pathophysiological mechanisms are involved in insulin resistance. Insulin resistance is a consequence of a complex combination of metabolic disorders, lipotoxicity, glucotoxicity, and inflammation. There is ample evidence linking different mechanistic approaches as the cause of insulin resistance, but no central mechanism is yet described as an underlying reason behind this condition. This review combines and interlinks the defects in the insulin signal transduction pathway of the insulin resistance state with special emphasis on the AGE-RAGE-NF-κB axis. Here, we describe important factors that play a crucial role in the pathogenesis of insulin resistance to provide directionality for the events. The interplay of inflammation and oxidative stress that leads to β-cell decline through the IAPP-RAGE induced β-cell toxicity is also addressed. Overall, by generating a comprehensive overview of the plethora of mechanisms involved in insulin resistance, we focus on the establishment of unifying mechanisms to provide new insights for the future interventions of type 2 diabetes mellitus.     

Hexokinase-2-Linked Glycolytic Overload and Unscheduled Glycolysis—Driver of Insulin Resistance and Development of Vascular Complications of Diabetes

The recent discovery of the glucose-induced stabilization of hexokinase-2 (HK2) to proteolysis in cell dysfunction in model hyperglycemia has revealed a likely key initiating factor contributing to the development of insulin resistance and vascular complications in diabetes. Consequently, the increased flux of glucose metabolism without a change in the expression and activity of glycolytic enzymes produces a wave of increased glycolytic intermediates driving mitochondrial dysfunction and increased reactive oxygen species (ROS) formation, the activation of hexosamine and protein kinase C pathways, the increased formation of methylglyoxal-producing dicarbonyl stress, and the activation of the unfolded protein response. This is called HK2-linked glycolytic overload and unscheduled glycolysis. The conditions required to sustain this are GLUT1 and/or GLUT3 glucose uptake and the expression of HK2. A metabolic biomarker of its occurrence is the abnormally increased deposition of glycogen, which is produced by metabolic channeling when HK2 becomes detached from mitochondria. These conditions and metabolic consequences are found in the vasculature, kidneys, retina, peripheral nerves, and early-stage embryo development in diabetes and likely sustain the development of diabetic vascular complications and embryopathy. In insulin resistance, HK2-linked unscheduled glycolysis may also be established in skeletal muscle and adipose tissue. This may explain the increased glucose disposal by skeletal uptake in the fasting phase in patients with type 2 diabetes mellitus, compared to healthy controls, and the presence of insulin resistance in patients with type 1 diabetes mellitus. Importantly, glyoxalase 1 inducer—trans-resveratrol and hesperetin in combination (tRES-HESP)—corrected HK2-linked glycolytic overload and unscheduled glycolysis and reversed insulin resistance and improved vascular inflammation in overweight and obese subjects in clinical trial. Further studies are now required to evaluate tRES-HESP for the prevention and reversal of early-stage type 2 diabetes and for the treatment of the vascular complications of diabetes.     

混合气中氯化氢过氯的早期发现

介绍了一种在合成氯乙烯工序的混合气(乙炔与氯化氢)中氯化氢微量过氯的早期发现方法,结合生产情况阐述了过氯发现过程、有效性及应用措施。     

Protein-Metabolite Interactions: Discovery and Significance

Metabolites orchestrate cellular processes as either substrates, co-enzymes, inhibitors, or activators of cellular proteins such as enzymes and receptors. Although traditional biochemical and structural biology-based approaches have been successfully employed for the discovery of protein-metabolite interactions, they often fail to detect transient and low-affinity biomolecular relationships. Another limitation of these approaches is that they are performed under in vitro conditions lacking the physiological context. Recently developed mass spectrometry-based methodologies overcome both these shortcomings, and have resulted in the discovery of global protein-metabolite cellular interaction networks. Herein, we describe traditional and modern approaches for the discovery of protein-metabolite interactions, and discuss the impact of these discoveries on our understanding of cellular physiology and on drug development.     

Advances in Oxidation Catalysis; Oxidation of Benzene to Phenol by Nutrous Oxide

Recent decades show impressive progress in oxidation catalysis, resulting in many novel industrial technologies. However, there is a difficult and challenging field, that of oxidation reactions which cannot be selectively implemented via traditional approaches. In the present paper, we discuss recent progress achieved in this particular field through the discovery of unique oxidation chemistry involving nitrous oxide.     

Activity-Directed Synthesis: A Flexible Approach for Lead Generation

Activity-directed synthesis (ADS) is a structure-blind, functional-driven molecular discovery approach. In this Concept, four case studies highlight the general applicability of ADS and showcase its flexibility to support different medicinal chemistry strategies. ADS deliberately harnesses reactions with multiple possible outcomes, and allows many chemotypes to be evaluated in parallel. Resources are focused on bioactive molecules, which emerge in tandem with associated synthetic routes. Some of the future challenges for ADS are highlighted, including the realisation of an autonomous molecular discovery platform. The prospects for ADS to become a mainstream lead generation approach are discussed.     

Structure‐Based and Fragment‐Based GPCR Drug Discovery

G protein‐coupled receptors (GPCRs) are an important family of membrane proteins; historically, drug discovery in this target class has been fruitful, with many of the world’s top‐selling drugs being GPCR modulators. Until recently, the modern techniques of structure‐ and fragment‐based drug discovery had not been fully applied to GPCRs, primarily because of the instability of these proteins when isolated from their cell membrane environments. Recent advances in receptor stabilisation have facilitated major advances in GPCR structural biology over the past six years, with 21 new receptor targets successfully crystallised with one or more ligands. The dramatic increase in GPCR structural information has yielded an increased use of structure‐based methods for hit identification and progression, which are reviewed herein. Additionally, a number of fragment‐based drug discovery techniques have been validated for use with GPCRs in recent years; these approaches and their use in hit identification are reviewed.     

Recent Advances on the Selection Methods of DNA-Encoded Libraries

DNA-encoded libraries (DEL) have come of age and become a major technology platform for ligand discovery in both academia and the pharmaceutical industry. Technological maturation in the past two decades and the recent explosive developments of DEL-compatible chemistries have greatly improved the chemical diversity of DELs and fueled its applications in drug discovery. A relatively less-covered aspect of DELs is the selection method. Typically, DEL selection is considered as a binding assay and the selection is conducted with purified protein targets immobilized on a matrix, and the binders are separated from the non-binding background via physical washes. However, the recent innovations in DEL selection methods have not only expanded the target scope of DELs, but also revealed the potential of the DEL technology as a powerful tool in exploring fundamental biology. In this Review, we first cover the “classic” DEL selection methods with purified proteins on solid phase, and then we discuss the strategies to realize DEL selections in solution phase. Finally, we focus on the emerging approaches for DELs to interrogate complex biological targets.