Developing hybrid molecule therapeutics for diverse enzyme inhibitory action: Active role of coumarin-based structural leads in drug discovery

Hybrid drugs featuring two or more potentially bioactive pharmacophores have been recognized as advanced and superior chemical entities to simultaneously modulate multiple drug targets of multifactorial diseases, thus overcoming the severe side effects associated with a single drug molecule. The selection of these chemical moieties to produce hybrid structures with druggable properties is generally facilitated by the observed and/or anticipated synergistic pharmacological activities of the individual molecules. In this perspective, coumarin template has extensively been studied in pursuit of structurally diverse leads for drug development due to high affinity and specificity to different molecular targets. This review highlights the most commonly exploited approaches conceptualizing the design and construction of hybrid molecules by coupling two or more individual fragments with or without an appropriate linker. In addition to the design strategies, this review also summarizes and reflects on the therapeutic potential of these hybrid molecules for diverse enzyme inhibitory action as well as their observed structure-activity relationship (SAR). Several key features of the synthesized hybrid structures that assert a profound impact on the inhibitory function have also been discussed alongside computational investigations, inhibitor molecular diversity and selectivity toward multiple drug targets. Finally, these drug discovery and development efforts should serve as a handy reference aiming to provide a useful platform for the exploration of new coumarin-based compounds with enhanced enzyme inhibitory profile.     

Oligonucleotides and derivatives as gene-specific control agents

The current achievement of genome sequence projects of a dozen eukaryote organisms (including human genome) and the development of functional genomics are providing the basic knowledge required to utilize gene-specific reagents for both basic understanding of cell physiology and therapeutical development. The field of chemical genomics has the ambitious goal of designing molecules that could act selectively on every single gene or gene product in a cell and in vivo. The progress in oligonucleotide-based approaches will be the topic of this review, however, other nucleic acid- and SELEX-based approaches as well as high sequence-specific low molecular weight DNA-specific ligands will also be discussed.     

Oligonucleotides and Derivatives as Gene-Specific Control Agents

Abstract

The current achievement of genome sequence projects of a dozen eukaryote organisms (including human genome) and the development of functional genomics are providing the basic knowledge required to utilize gene-specific reagents for both basic understanding of cell physiology and therapeutical development. The field of chemical genomics has the ambitious goal of designing molecules that could act selectively on every single gene or gene product in a cell and in vivo. The progress in oligonucleotide-based approaches will be the topic of this review, however, other nucleic acid- and SELEX-based approaches as well as high sequence-specific low molecular weight DNA-specific ligands will also be discussed.     

Signaling protein networks as targets of new antineoplastic drugs

In-depth analysis of molecular regulatory networks in cancer holds the promise of improved knowledge of the pathophysiology of tumor cells so that it will become possible to design a detailed molecular tumor taxonomy. This knowledge will also offer new opportunities for the identification and validation of key molecular tumor targets to be exploited for novel therapeutic approaches. Some signaling proteins have already been identified as such, e.g. c-Myc, Cyclin D1, Bcl-XL, kinases and some nuclear receptors. This has led to the successful development of a few function-modulatory drugs (Glivec, SERM, Iressa), providing proof-of-principle of the validity of this approach. Further developments are likely to derive from "-omic" approaches, aimed at the understanding of signaling networks and of the mechanism of action of newfound lead molecules. High-throughput screening of small drug-like molecules from combinatorial chemical libraries or from microbial extracts will identify novel, "intelligent" drug candidates. An additional medicinal chemistry strategy (via 40-50 unit rosary-bead chains) has the potential to be much more effective than small molecules in interfering with protein-protein interactions. This may lead to considerably higher selectivity and effectiveness compared with historical approaches in drug discovery.     

Structural genomics for Caenorhabditis elegans: high throughput protein expression analysis

The structural genomics initiatives have begun with the aim to create a so-called "basic set library" of protein folds that will be used to improve protein prediction methods. Such a library is thought to require the determination of up to 10,000 new structures, including representative structures of several sequence variants from each protein fold. To meet this goal in a reasonable time frame and cost, automated systems must be utilized to clone and to identify the soluble recombinant proteins contained in multiple genomes. This paper presents such a system, developed using the genome of Caenorhabditis elegans (19,099 genes) as a model eukaryotic organism for structural genomics. This system successfully automates nearly all aspects of recombinant protein expression analysis including subcloning, bacterial growth, recombinant protein expression, protein purification, and scoring protein solubility.     

酵母双杂合系统的改进和发展

酵母双杂合系统是在１９８９年由ＳｔａｎｌｅｙＦｉｅｌｄｓ和Ｏｋ－ｋｙｕＳｏｎｇ等提出并初步建立的［１］,该系统是在酿酒酵母（Ｓａｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）中研究蛋白质间相互作用的一种非常有效的分子生物学方法。近几年来随着人们对该系统的广泛应用,这一系统得到了不断的完善及改进,同时也衍生出单杂合系统,三杂合系统等一系列相关的技术。这些技术在不同研究领域中的广泛应用有力地推动了蛋白质与ＤＮＡ,蛋白质与ＲＮＡ,以及多种蛋白质分子间相互作用的研究。     

Rational approaches,design strategies,structure activity relationship and mechanistic insights for therapeutic coumarin hybrids

Hybrid molecules, furnished by combining two or more pharmacophores is an emerging concept in the field of medicinal chemistry and drug discovery that has attracted substantial traction in the past few years. Naturally occurring scaffolds such as coumarins display a wide spectrum of pharmacological activities including anticancer, antibiotic, antidiabetic and others, by acting on multiple targets. In this view, various coumarin-based hybrids possessing diverse medicinal attributes were synthesized in the last five years by conjugating coumarin moiety with other therapeutic pharmacophores. The current review summarizes the recent development (2014 and onwards) of these pharmacologically active coumarin hybrids and demonstrates rationale behind their design, structure-activity relationships (SAR) and mechanistic studies performed on these hybrid molecules. This review will be beneficial for medicinal chemist and chemical biologist, and in general to the drug discovery community and will facilitate the synthesis and development of novel, potent coumarin hybrid molecules serving as lead molecules for the treatment of complex disorders.     

Peptides as protein binding site mimetics

The rational/structure-based design and/or combinatorial development of molecules capable of structurally and functionally mimicking the binding sites of proteins represents a promising strategy for the exploration and understanding of protein structure and function. The ultimate goal of using such molecules is the modulation of protein function through controlled interference with the underlying binding events. In addition to their basic significance, such proteinmimetics are also useful tools for a range of biomedical applications, in particular the inhibition of disease-associated protein-ligand interactions. Owing to their chemical and structural relation to proteins, as well as the relative simplicity of their chemical or recombinant synthesis, peptides have emerged as adequate molecules for the mimicry of protein binding sites, as well as the inhibition of protein-protein interactions.     

Chemical genomics and medicinal systems biology: chemical control of genomic networks in human systems biology for innovative medicine

With advances in determining the entire DNA sequence of the human genome, it is now critical to systematically identify the function of a number of genes in the human genome. These biological challenges, especially those in human diseases, should be addressed in human cells in which conventional (e.g. genetic) approaches have been extremely difficult to implement. To overcome this, several approaches have been initiated. This review will focus on the development of a novel "chemical genetic/genomic approach" that uses small molecules to "probe and identify" the function of genes in specific biological processes or pathways in human cells. Due to the close relationship of small molecules with drugs, these systematic and integrative studies will lead to the "medicinal systems biology approach" which is critical to "formulate and modulate" complex biological (disease) networks by small molecules (drugs) in human bio-systems.     

What does it take to make a heart?

Ever increasing advances are being made in our quest to understand what it takes to direct pluripotent precursor cells to adopt a specific developmental fate. Eventually, the obvious goal is that targeted manipulation of these precursor cells will result in an efficient and reliable production of tissue‐specific cells, which can be safely employed for therapeutic purposes. We have gained an incredible insight as to which molecular pathways are involved in governing neural, skeletal and cardiac muscle fate decisions. However, we still face the challenge of how to direct, for example, a cardiac fate in stem cells in the amounts needed to be employed for regenerative means. Equally importantly, we need to resolve critical questions such as: can the in vitro generated cardiomyocytes actually functionally replace damaged heart tissue? Here I will provide an overview of the molecules and signalling pathways that have first been demonstrated in embryological studies to function in cardiogenesis, and summarize how this knowledge is being applied to differentiate mouse and human embryonic stem cells into cardiomyocytes.     

Enzyme overproduction: Principle and application

The rapprochement between gene physiology and protein chemistry proffered a wishful manipulation or programming of biological blueprint which we now know as recombinant DNA technology. Its premises are very many and ends are manifold. Until rather recently, the recombinant DNA technique could conceive the idea of engineering enzyme molecules by cloning and selection of the gene in question for enzyme production. At least, in principle, the process is too simple but its underlying mechanism is rather much stringent. Various experimental paradigms have been brought to work for production of enzyme at will by introducing a given gene into a high yielding system of microorganisms. It facilitates overproduction of enzymes of interest which can be implicated in several important industrial, biomedical, and environmental processes at a large scale. Such approaches of enzymes made-to-application have already started asserting tremendously in doing their appropriate jobs at the level of molecular interactions. A rapid progress in this important and interesting area of biocatalytic manipulation will certainly achieve the goal of biocatalysis-made-to-order by altering kinetic and thermodynamic components of enzyme molecules.     

Synthetic Approach to biomolecular science by cyborg supramolecular chemistry

Background

To imitate the essence of living systems via synthetic chemistry approaches has been attempted. With the progress in supramolecular chemistry, it has become possible to synthesize molecules of a size and complexity close to those of biomacromolecules. Recently, the combination of precisely designed supramolecules with biomolecules has generated structural platforms for designing and creating unique molecular systems. Bridging between synthetic chemistry and biomolecular science is also developing methodologies for the creation of artificial cellular systems.

Macromolecular therapeutics: emerging strategies for drug discovery in the postgenome era

The postgenome era offers a plethora of potential therapeutic targets. Many of these targets will be addressable using small organic molecules as drug candidates. However, certain aspects of cell function, particularly those that rely on protein-protein or protein-nucleic acid interactions, will be difficult to influence using small molecules. Thus, the possibility of using highly specific macromolecules as potential therapeutic agents is an intriguing concept. Recent developments in several areas of research have brought this possibility closer to fruition. Peptide and nucleic acid combinatorial libraries allow the generation of novel molecules having exquisite selectivity. Structural information and molecular modeling also contribute to the design of new macromolecules with therapeutic potential. Perhaps most importantly, approaches for delivering macromolecules into the cell interior have been developed and applied with considerable success. Thus, the therapeutic use of macromolecules, including oligonucleotides, peptides, and proteins, may be an idea whose time has come.     

A metalloantibody that irreversibly binds a protein antigen

Antibody affinity is critically important in therapeutic applications, as well as steady state diagnostic assays. Picomolar affinity antibodies, approaching the association limit of protein-protein interactions, have been discovered for highly potent antigens, but even such high-affinity binders have off-rates sufficient to negate therapeutic efficacy. To cross this affinity threshold, antibodies that tether their targets in a manner other than reversible non-covalent interaction will be required. Here we report the design and construction of an antibody that forms an irreversible complex with a protein antigen in a metal-dependent reaction. The complex resists thermal and chemical denaturation, as well as attempts to remove the coordinating metal ion. Such irreversibly binding antibodies could facilitate the development of next generation "reactive antibody" therapeutics and diagnostics.     

Antibody conjugates and therapeutic strategies

Immunotherapeutics represent the largest group of molecules currently in development as new drug entities. These versatile molecules are being investigated for the treatment of a range of pathological conditions including cancer, infectious and inflammatory diseases. Antibodies can be used to exert biological effects themselves or as delivery agents of conjugated drug molecules. Site-specific delivery of therapeutic agents has been an ultimate goal of the pharmaceutical industry in order to maximize drug action and minimize side effects. Antibodies have the potential to realize this objective and in this review we will examine some of the main strategies currently being employed for the development of these diverse therapeutic molecules.     

Hydrocortisone made in yeast: metabolic engineering turns a unicellular microorganism into a drug-synthesizing factory

Inspired by the successful work of converting Saccharomyces cerevisiae into an microorganism capable of synthesizing hydrocortisone, a 27-carbon molecule, from ethanol, a 2-carbon molecule, this review provides an overview of the potential of yeast as a recombinant organism in the 21st century. Yeast has been used by man for more than 6,000 years, and is still paving the way to new discoveries. It was the first eukaryotic organism to be sequenced, in 1996, and the first to produce hydrocortisone in 2003. In addition, extensive genome-wide analyses have been performed with yeast. In this review, we discuss the pros and cons of using yeast to produce small therapeutic molecules. It is obvious that S. cerevisiae has a cutting edge advantage of being a well-known organism and time will tell if yeast "biohydrocortisone" is a unique example or the beginning of a long list of yeast bioproducts. Other organisms, such as plants and bacteria, are competing with yeast. Bacteria produce a wealth of marketed molecules and plants are capable of producing extremely complex molecules with an unbeatable yield. However, S. cerevisiae offers a unique mix of the simplicity of a recombinant organism combined with the complexity of a eukaryote.     

Chemical approaches to the lysophospholipid receptors

Both ligand-based and GPCR privileged scaffold chemical tools have recently emerged to provide new insights into the function and physiology of the GPCR lysophospholipid receptors both in vitro and in vivo. Both rational, design-based approaches as well as hybrid approaches where high throughput screening has been coupled to an understanding of critical molecular interactions have been productive in advancing understanding of physiology and potential therapeutics in this field. It is now feasible to identify reasonably potent and selective small molecules that provide chemical proof-of-concept in vivo directly from high throughput screening. These developments, coupled with the availability of receptor knock-out mice, presage rapid progress in the field.     

Secretome proteomics for discovery of cancer biomarkers

“Secretome” is referred to as the rich, complex set of molecules secreted from living cells. In a less strict definition frequently followed in “secretome” studies, the term also includes molecules shed from the surface of living cells. Proteins of secretome (will be referred to as secreted) play a key role in cell signaling, communication and migration. The need for developing more effective cancer biomarkers and therapeutic modalities has led to the study of cancer cell secretome as a means to identify and characterize diagnostic and prognostic markers and potential drug and therapeutic targets. Significant technological advances in the field of proteomics during the last two decades have greatly facilitated research towards this direction. Nevertheless, secretome analysis still faces some difficulties mainly related to sample collection and preparation. The goal of this article is to provide an overview of the main findings from the analysis of cancer cell secretome. Specifically, we focus on the presentation of main methodological approaches that have been developed for the study of secreted proteins and the results thereof from the analysis of secretome in different types of malignancies; special emphasis is given on correlation of findings with protein expression in body fluids.     

Teaching an Old Virus New Tricks: A Review on New Approaches to Study Age-Old Questions in Influenza Biology

Influenza viruses have been studied for over 80 years, yet much about the basic viral lifecycle remain unknown. However, new imaging, biochemical, and sequencing techniques have revealed significant insight into many age-old questions of influenza virus biology. In this review, we will cover the role of imaging techniques to describe unique aspects of influenza virus assembly, biochemical techniques to study viral genomic organization, and next-generation sequencing to explore influenza genomic evolution. Our goal is to provide a brief overview of how emerging techniques are being used to answer basic questions about influenza viruses. This is not a comprehensive list of emerging techniques, rather ones that we feel will continue to make significant contributions to field of influenza biology.     

Nonviral gene therapy targeting cardiovascular system

The goal of gene therapy is either to introduce a therapeutic gene into or replace a defective gene in an individual's cells and tissues. Gene therapy has been urged as a potential method to induce therapeutic angiogenesis in ischemic myocardium and peripheral tissues after extensive investigation in recent preclinical and clinical studies. A successful gene therapy mainly relies on the development of the gene delivery vector. Developments in viral and nonviral vector technology including cell-based gene transfer will further improve transgene delivery and expression efficiency. Nonviral approaches as alternative gene delivery vehicles to viral vectors have received significant attention. Recently, a simple and safe approach of gene delivery into target cells using naked DNA has been improved by combining several techniques. Among the physical approaches, ultrasonic microbubble gene delivery, with its high safety profile, low costs, and repeatable applicability, can increase the permeability of cell membrane to macromolecules such as plasmid DNA by its bioeffects and can provide as a feasible tool in gene delivery. On the other hand, among the promising areas for gene therapy in acquired diseases, ischemic cardiovascular diseases have been widely studied. As a result, gene therapy using advanced technology may play an important role in this regard. The aims of this review focus on understanding the cellular and in vivo barriers in gene transfer and provide an overview of currently used chemical vectors and physical tools that are applied in nonviral cardiovascular gene transfer.