Interaction between Melatonin and NO: Action Mechanisms,Main Targets,and Putative Roles of the Emerging Molecule NOmela

Melatonin (MEL), a ubiquitous indolamine molecule, has gained interest in the last few decades due to its regulatory role in plant metabolism. Likewise, nitric oxide (NO), a gasotransmitter, can also affect plant molecular pathways due to its function as a signaling molecule. Both MEL and NO can interact at multiple levels under abiotic stress, starting with their own biosynthetic pathways and inducing a particular signaling response in plants. Moreover, their interaction can result in the formation of NOmela, a very recently discovered nitrosated form of MEL with promising roles in plant physiology. This review summarizes the role of NO and MEL molecules during plant development and fruit ripening, as well as their interactions. Due to the impact of climate-change-related abiotic stresses on agriculture, this review also focuses on the role of these molecules in mediating abiotic stress tolerance and the main mechanisms by which they operate, from the upregulation of the entire antioxidant defense system to the post-translational modifications (PTMs) of important molecules. Their individual interaction and crosstalk with phytohormones and H₂S are also discussed. Finally, we introduce and summarize the little information available about NOmela, an emerging and still very unknown molecule, but that seems to have a stronger potential than MEL and NO separately in mediating plant stress response.     

Toxin-Induced Experimental Models of Learning and Memory Impairment

Animal models for learning and memory have significantly contributed to novel strategies for drug development and hence are an imperative part in the assessment of therapeutics. Learning and memory involve different stages including acquisition, consolidation, and retrieval and each stage can be characterized using specific toxin. Recent studies have postulated the molecular basis of these processes and have also demonstrated many signaling molecules that are involved in several stages of memory. Most insights into learning and memory impairment and to develop a novel compound stems from the investigations performed in experimental models, especially those produced by neurotoxins models. Several toxins have been utilized based on their mechanism of action for learning and memory impairment such as scopolamine, streptozotocin, quinolinic acid, and domoic acid. Further, some toxins like 6-hydroxy dopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and amyloid-β are known to cause specific learning and memory impairment which imitate the disease pathology of Parkinson’s disease dementia and Alzheimer’s disease dementia. Apart from these toxins, several other toxins come under a miscellaneous category like an environmental pollutant, snake venoms, botulinum, and lipopolysaccharide. This review will focus on the various classes of neurotoxin models for learning and memory impairment with their specific mechanism of action that could assist the process of drug discovery and development for dementia and cognitive disorders.     

Fluorescent Mechanism‐Based Probe for Aerobic Flavin‐Dependent Enzyme Activity

Diversity in non‐ribosomal peptide and polyketide secondary metabolism is facilitated by interactions between biosynthetic domains with discrete monomer loading and their cognate tailoring enzymes, such as oxidation or halogenation enzymes. The cooperation between peptidyl carrier proteins and flavin‐dependent enzymes offers a specialized strategy for monomer selectivity for oxidization of small molecules from within a complex cellular milieu. In an effort to study this process, we have developed fluorescent probes to selectively label aerobic flavin‐dependent enzymes. Here we report the preparation and implementation of these tools to label oxidase, monooxygenase, and halogenase flavin‐dependent enzymes.     

Establishing a new methodology for genome mining and biosynthesis of polyketides and peptides through yeast molecular genetics

Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (5-20 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.     

Fusion Enzymes Involved in Biosynthetic Tailoring Reactions in Fungi

Enzyme fusion, the fusion of enzymes with different domains to a single protein, has been widely recognized as a promising strategy in the development of biocatalysts. Nature has evolved gene fusion to combine different catalytic enzymes to function as a fusion enzyme, and this strategy is utilized in many natural product biosynthetic pathways. Owing to rapid advances in genome sequencing and biosynthetic pathway characterization, there is increasing interest in fusion enzymes from fungal biosynthetic pathways, particularly those involved in tailoring steps. This concept aims to provide an up-to-date overview of fusion enzymes that catalyze tailoring reactions in the biosynthesis of fungal secondary metabolites. Since fungal fusion enzymes are often associated with novel metabolites, this pioneering work may stimulate the exploration of the structural diversity of fungal natural products through genome mining of the untapped biosynthetic pathways involving fusion enzymes.     

Combinatorial biosynthesis of antibiotics: challenges and opportunities

Natural products with antibiotic activity have been central agents in human therapeutics over the past fifty years. They are likely to remain crucial in the decades to come. These molecules, often termed secondary metabolites because they are the end products of dedicated metabolic pathways that are turned on when microbes are stressed by environmental factors such as starvation, can achieve considerable architectural and functional group complexity that allows specific targeting. The programmed manipulation of the genes that encode the enzymes in the biosynthetic pathways offers promise for redesign of antibiotic structures to create new activities and overcome bacterial resistance to existing antibiotics.     

Identification and Characterization of the Streptazone E Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08

Streptazone derivatives isolated from Streptomyces species are piperidine alkaloids with a cyclopenta[b]pyridine scaffold. Previous studies indicated that these compounds are polyketides, but the biosynthetic enzymes responsible for their synthesis are unknown. Here, we have identified the streptazone E biosynthetic gene cluster in Streptomyces sp. MSC090213JE08, which encodes a modular type I PKS and tailoring enzymes that include an aminotransferase, three oxidoreductases, and two putative cyclases. The functions of the six tailoring enzymes were analyzed by gene disruption, and two putative biosynthetic intermediates that accumulated in particular mutants were structurally elucidated. On the basis of these results, we propose a pathway for the biosynthesis of streptazone E in which the two putative cyclases of the nuclear transport factor 2–like superfamily are responsible for C?C bond formation coupled with epoxide ring opening to give the five‐membered ring of streptazone E.     

Uremic Toxins and Their Relation with Oxidative Stress Induced in Patients with CKD

The presence of toxins is believed to be a major factor in the development of uremia in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD). Uremic toxins have been divided into 3 groups: small substances dissolved in water, medium molecules: peptides and low molecular weight proteins, and protein-bound toxins. One of the earliest known toxins is urea, the concentration of which was considered negligible in CKD patients. However, subsequent studies have shown that it can lead to increased production of reactive oxygen species (ROS), and induce insulin resistance in vitro and in vivo, as well as cause carbamylation of proteins, peptides, and amino acids. Other uremic toxins and their participation in the damage caused by oxidative stress to biological material are also presented. Macromolecules and molecules modified as a result of carbamylation, oxidative stress, and their adducts with uremic toxins, may lead to cardiovascular diseases, and increased risk of mortality in patients with CKD.     

The Discovery of New Cyanobactins from Cyanothece PCC 7425 Defines a New Signature for Processing of Patellamides

Cyanobactins, including patellamides, are a group of cyanobacterial post‐translationally modified ribosomal cyclic peptides. The final product should in theory be predictable from the sequence of the precursor peptide and the associated tailoring enzymes. Understanding the mechanism and recognition requirements of these enzymes will allow their rational engineering. We have identified three new cyanobactins from a Cyanothece PCC 7425 culture subjected to a heat shock. One of these compounds revealed a novel signature signal for ThcA, the subtilisin‐like serine protease that is homologous to the patellamide protease PatA. The crystal structure of the latter and modelling studies allow rationalisation of the recognition determinants for both enzymes, consistent with the ribosomal biosynthetic origin of the new compounds.     

Targeting Cancer Stem Cells with Small Molecules

Cancers arise as a result of physiological imbalances and subsequent uncontrolled cell division. Cancer initiation requires a set of biochemical alterations, including some occurring at the genetic and epigenetic levels. Thus, tumors are heterogeneous in nature making it challenging to selectively target different cancer cells by means of small molecule intervention. The paradigm of cancer stem cells (CSCs) describes subpopulations of cells with high self-renewal and tumor-seeding capacity. These cells, typically refractory to conventional therapies, can give rise to relapse after treatment. Combinatorial strategies, including drugs that selectively target this population of cells, have emerged in recent years. Here, we review how discovery-based – unbiased – screening approaches 1 have helped identify small molecules that specifically target CSCs. We also highlight biological pathways characteristic of CSCs that can potentially be selectively targeted in a hypothesis-driven manner by small molecules. We describe molecules that effectively target CSCs and emphasize what is known about their biological modes of action. The diversity and complexity of biochemical processes that CSCs may be addicted to, raises the question of how selective targeting of these pathways can be achieved. This challenge may be addressed by the continuing production of structurally complex and diverse small molecules using target and diversity-oriented synthesis approaches. 2     

Purification and Kinetic Characterization of the Essential Condensation Enzymes Involved in Prodiginine and Tambjamine Biosynthesis

Prodiginines and tambjamines are related families of bioactive alkaloid natural products with pharmaceutical potential. Both compound families result from a convergent biosynthetic pathway ending in the condensation of a conserved bipyrrole core with a variable partner. This reaction is performed by unique condensation enzymes, and has the potential to be manipulated to produce new pyrrolic compounds. We have purified and reconstituted the in vitro activity of the condensation enzymes PigC and TamQ from Pseudoalteromonas sp., which are involved, respectively, in the prodiginine and tambjamine biosynthetic pathways. Kinetic analysis confirmed a Uni Uni Bi Uni ping-pong reaction sequence with competitive and uncompetitive substrate inhibition for PigC and TamQ respectively. The kinetic parameters of each enzyme provide insight into their differing substrate scope, and suggest that TamQ may have evolved a wide substrate tolerance that can be used for the production of novel prodiginines and tambjamines.     

Molecular Genetics of Plant Sterol Backbone Synthesis

Sterols, which are biosynthesized via the cytoplasmic mevalonate (MVA) pathway, are important structural components of the plasma membrane and precursors of steroid hormones in both vertebrates and plants. Ergosterol and cholesterol are the major sterols in yeast and vertebrates, respectively. In contrast, plants produce a wide variety of phytosterols, which have various functions in plant development. Although the general biosynthetic pathway to plant sterols has been defined, the details of the biochemical, physiological, and developmental functions of genes involved in the biosynthetic network and their regulation are not well understood. Molecular genetic analyses are an effective approach to use when studying these fascinating problems. Since three enzymes, 3-hydroxy-3-methylglutaryl CoA reductase, farnesyl diphosphate synthase, and lanosterol synthase, have been functionally characterized in planta, we reviewed recent progress on these enzymes. Arabidopsis T-DNA and transposon insertion mutants are now widely available. The use of molecular genetics, molecular biology, and bioorganic chemical approaches on these mutants, as well as inhibitors of the MVA pathway, should help us to understand plant sterol biosynthesis comprehensively.     

Identification of small-molecule inhibitors of the XendoU endoribonucleases family

The XendoU family of enzymes includes several proteins displaying high sequence homology. The members characterized so far are endoribonucleases sharing similar biochemical properties and a common architecture in their active sites. Despite their similarities, these proteins are involved in distinct RNA-processing pathways in different organisms. The amphibian XendoU participates in the biosynthesis of small nucleolar RNAs, the human PP11 is supposed to play specialized roles in placental tissue, and NendoU has critical function in coronavirus replication. Notably, XendoU family members have been implicated in human pathologies such as cancer and respiratory diseases: PP11 is aberrantly expressed in various tumors, while NendoU activity has been associated with respiratory infections by pathogenic coronaviruses. The present study is aimed at identifying small molecules that may selectively interfere with these enzymatic activities. Combining structure-based virtual screening and experimental approaches, we identified four molecules that specifically inhibited the catalytic activity of XendoU and PP11 in the low micromolar range. Moreover, docking experiments strongly suggested that these compounds might also bind to the active site of NendoU, thus impairing the catalytic activity essential for the coronavirus life cycle. The identified compounds, while allowing deep investigation of the molecular functions of this enzyme family, may also represent leads for the development of new therapeutic tools.     

Probing the compatibility of type II ketosynthase-carrier protein partners

Drug discovery often begins with the screening of large compound libraries to identify lead compounds. Recently, the enzymes that are involved in the biosynthesis of natural products have been investigated for their potential to generate new, diverse compound libraries. There have been several approaches toward this end, including altering the substrate specificities of the enzymes involved in natural product biosynthesis and engineering functional communication between enzymes from different biosynthetic pathways. While there exist assays to assess the substrate specificity of enzymes involved in these pathways, there is no simple method for determining whether enzymes from different synthases will function cooperatively to generate the desired product(s). Herein we report a method that provides insight into both substrate specificity and compatibility of protein-protein interactions between the acyl carrier protein (ACP) and ketosynthase (KS) domains involved in fatty acid and polyketide biosynthesis. Our technique uses a one-pot chemoenzymatic method to generate post-translationally modified ACPs that are capable of covalently interacting with KS domains from different biosynthetic systems. The extent of interaction between ACPs and KSs from different systems is easily detected and quantified by a gel-based method. Our results are consistent with previous studies of substrate specificity and ACP-KS binding interactions and provide new insight into unnatural substrate and protein interactions.     

Examination of Secondary Metabolite Biosynthesis in Apicomplexa

Natural product discovery has traditionally relied on the isolation of small molecules from producing species, but genome-sequencing technology and advances in molecular biology techniques have expanded efforts to a wider array of organisms. Protists represent an underexplored kingdom for specialized metabolite searches despite bioinformatic analysis that suggests they harbor distinct biologically active small molecules. Specifically, pathogenic apicomplexan parasites, responsible for billions of global infections, have been found to possess multiple biosynthetic gene clusters, which hints at their capacity to produce polyketide metabolites. Biochemical studies have revealed unique features of apicomplexan polyketide synthases, but to date, the identity and function of the polyketides synthesized by these megaenzymes remains unknown. Herein, we discuss the potential for specialized metabolite production in protists and the possible evolution of polyketide biosynthetic gene clusters in apicomplexan parasites. We then focus on a polyketide synthase from the apicomplexan Toxoplasma gondii to discuss the unique domain architecture and properties of these proteins when compared to previously characterized systems, and further speculate on the possible functions for polyketides in these pathogenic parasites.     

Transient Receptor Potential Channels and Itch

Transient Receptor Potential (TRP) channels are multifunctional sensory molecules that are abundant in the skin and are involved in the sensory pathways of itch, pain, and inflammation. In this review article, we explore the complex physiology of different TRP channels, their role in modulating itch sensation, and their contributions to the pathophysiology of acute and chronic itch conditions. We also cover small molecule and topical TRP channel agents that are emerging as potential anti-pruritic treatments; some of which have shown great promise, with a few treatments advancing into clinical trials—namely, TRPV1, TRPV3, TRPA1, and TRPM8 targets. Lastly, we touch on possible ethnic differences in TRP channel genetic polymorphisms and how this may affect treatment response to TRP channel targets. Further controlled studies on the safety and efficacy of these emerging treatments is needed before clinical use.     

Biosynthesis and Genome Mining Potentials of Nucleoside Natural Products

Nucleoside natural products show diverse biological activities and serve as leads for various application purposes, including human and veterinary medicine and agriculture. Studies in the past decade revealed that these nucleosides are biosynthesized through divergent mechanisms, in which early steps of the pathways can be classified into two types (C5' oxidation and C5' radical extension), while the structural diversity is created by downstream tailoring enzymes. Based on this biosynthetic logic, we investigated the genome mining discovery potentials of these nucleosides using the two enzymes representing the two types of C5' modifications: LipL-type α-ketoglutarate (α-KG) and Fe-dependent oxygenases and NikJ-type radical S-adenosyl-L-methionine (SAM) enzymes. The results suggest that this approach allows discovery of putative nucleoside biosynthetic gene clusters (BGCs) and the prediction of the core nucleoside structures. The results also revealed the distribution of these pathways in nature and implied the possibility of future genome mining discovery of novel nucleoside natural products.