Drug design for ever, from hype to hope

In its first 25 years JCAMD has been disseminating a large number of techniques aimed at finding better medicines faster. These include genetic algorithms, COMFA, QSAR, structure based techniques, homology modelling, high throughput screening, combichem, and dozens more that were a hype in their time and that now are just a useful addition to the drug-designers toolbox. Despite massive efforts throughout academic and industrial drug design research departments, the number of FDA-approved new molecular entities per year stagnates, and the pharmaceutical industry is reorganising accordingly. The recent spate of industrial consolidations and the concomitant move towards outsourcing of research activities requires better integration of all activities along the chain from bench to bedside. The next 25 years will undoubtedly show a series of translational science activities that are aimed at a better communication between all parties involved, from quantum chemistry to bedside and from academia to industry. This will above all include understanding the underlying biological problem and optimal use of all available data

ELUCIDATION OF THE NEEDLE-TIP AND TIP-TRANSLOCON INTERACTIONS OF THE SALMONELLA SPI-1 TYPE III SECRETION SYSTEM AND IDENTIFICATION OF SMALL MOLECULE BINDERS OF THE TIP AND TRANSLOCON PROTEINS

The type III secretion system (T3SS) is required by many pathogenic Gram-negative bacteria for the initiation and maintenance of infections within eukaryotic host cells. T3SS harboring bacteria include the causative agents of food poisoning/typhoid fever (Salmonella Typhimurium/Typhi), dysentery (Shigella flexneri/dysenteriae), nosocomial pneumonia (Pseudomonas aeruginosa), bubonic plague (Yersinia pestis), melioidosis (Burkholderia pseudomallei), and trachoma (Chlamydia trachomatis). Together, these bacteria are estimated to result in millions of deaths worldwide each year. Therefore, it is of great interest to elucidate the mechanisms of T3SS-mediated virulence utilized by pathogenic Gram-negative bacteria. Salmonella is the focus of this dissertation because it is an excellent model organism for T3SS research due to the ease of genetic manipulation and the availability of biological assays.The T3SS is utilized to inject bacterial virulence factors (also known as effectors) into the host cell cytoplasm, where they manipulate host cell signaling pathways to promote bacterial engulfment, maintenance of infection, and evasion of the host immune system. T3SS effectors are translocated across both bacterial and host cell membranes by the structural component of the T3SS, the needle apparatus. The needle apparatus contains a bacterial membrane embedded base structure, an extracellular needle with a 25A wide channel, a tip complex that regulates secretion and serves as an environmental sensor, and translocon proteins that assemble a pore in the host cell membrane. How the needle, tip and translocon proteins interact with each other to assemble a functional T3SS needle apparatus and coordinate the secretion of T3SS effectors is poorly understood. Because the needle, tip and translocon proteins are essential for the pathogenesis of T3SS harboring bacteria, are exposed to the extracellular environment during infection, and are conserved in structure and function, they are attractive targets for the development of novel virulence based anti-bacterial therapeutics. Hence, the importance of elucidating the structure, function and molecular interactions of the T3SS needle, tip and translocon proteins.This dissertation is focused on two major themes. The first theme is the elucidation of essential protein-protein interactions of the Salmonella T3SS needle apparatus through a combination of solution nuclear magnetic resonance (NMR) and fluorescence spectroscopy. To this end, I used amide (15N) and isoleucine, leucine and valine methyl (ILV 13C-methyl) probes in NMR titrations to map the interaction of T3SS proteins. I additionally labeled T3SS proteins with fluorescent probes to perform fluorescence polarization (FP) and Förster resonance energy transfer (FRET) protein- protein binding assays to complement the NMR studies. Using these methods, the interaction between the Salmonella SP-1 T3SS needle protein PrgI and the tip protein SipD, as well as between SipD and the major translocon protein SipB, are described in detail and validated using bacterial invasion assays. The results of NMR and FP/FRET experiments allowed for the proposal of a model for the needle/tip/translocon protein- protein interaction interface where the proximal end of SipD (the bottom of the coiled- coil) is used for interaction with PrgI, while the distal end of SipD (the top of the coiled- coil and the mixed α/β domain) is the surface used for interaction with SipB. The second theme is focused on the T3SS needle apparatus as an attractive target for the development of inhibitors. A review of the current T3SS inhibitor literature is described. In addition, I identified small molecules binders of the tip and translocon proteins from a surfaceivplasmon resonance (SPR) screen and subsequently validated and mapped the protein- small molecule interactions using titration and saturation transfer different (STD) NMR spectroscopy

Characterization of antimicrobial peptides deriving from insects and their application in the biomedical field

Antibiotics are the current drugs used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms. The antibiotic resistance issue led to the need to find new alternative molecules, which should be less prone to bacterial resistance. Antimicrobial peptides (AMPs) aroused great interest as potential next-generation antibiotics. AMPs are involved in several defence-related processes such as the binding and neutralization of endotoxins, the modulation of the immune responses to infection and the killing of pathogens. Antimicrobial peptides are small molecules with an amino acid composition ranging from 10 to 100 residues and are biosynthesized by all living organisms but it is known that the class of insects represents the largest source of these molecules. This aspect is related to insect’s biodiversity and their ability to live in hostile environments rich of pathogens. Most insect AMPs are cationic molecules due to the presence of basic residues and according to their amino acid sequences and structures, they can be classified in four different groups: cysteine-rich peptides (e.g., defensins), the α-helical peptides (e.g., cecropins), glycine-rich proteins (e.g., attacins) and proline-rich peptides (e.g., drosocins). Insect AMPs have demonstrated to be useful in several applications concerning the pharmaceutical as well as the agricultural fields. Moreover, insect AMPs aroused great interest for their biomedical application thanks to the increasing number of peptides that can inhibit human pathogens. For this reason, this Ph.D. project aimed to the identification of antimicrobial peptides deriving from insects, particularly from the Black Soldier Fly Hermetia illucens (L.) (Diptera: Stratiomyidae). Through a combination of transcriptomics and bioinformatics analysis, 57 antimicrobial peptides have been identified from H. illucens insect. Through an in silico analysis, the biological activity have been predicted and the physio-chemical properties have been calculated for all the identified peptides. Based on the bioinformatics results, the in vitro production of the most promising sequences has been performed through molecular cloning strategies in order to evaluate the antibacterial activity in vitro. Particularly, some of the identified peptides (C16571, C46948, C16634, and C7985) showed the ability to inhibit E. coli growth at a concentration value of 3 μM. For the C15867 peptide, recombinantly produced and expressed, a MIC (Minimum Inhibitory Concentration) value of 18 μM has been determined. Moreover, an in vivo approach was carried out for the identification of antimicrobial peptides by extracting the hemolymph from the H. illucens larvae, recovering then the peptides fraction from the larvae’s plasma and its antibacterial activity has been evaluated against both Gram-positive and Gram-negative bacteria. The performed analysis showed that a small amount (7.5/15 μL) of the peptide fraction recovered from the larvae’s plasma was able to inhibit the cell growth of different bacterial strains

Antimicrobial Peptides Aka Host Defense Peptides – From Basic Research to Therapy

This Special Issue reprint will address the most current and innovative developments in the field of HDP research across a range of topics, such as structure and function analysis, modes of action, anti-microbial effects, cell and animal model systems, the discovery of novel host-defense peptides, and drug development

Nanochannels for electrical biosensing

This review shows the recent trends on the use of both single and array nanochannels for electrical biosensing applications. Some general considerations on the principles of the stochastic sensing, together with an overview about the common routes for nanochannels preparation before focusing on the applications for DNA, protein, virus, toxin and other analytes detection are given. Emerging materials used to obtain nanochannels, such as graphene and its analogues as well as novel systems based on the use of nanoparticles in combination with nanochannels are discussed. Aspects related to the analytical performance of the developed devices are also discussed. Finally prospects for future improvements and applications of this technology are included

Antibacterial Surfaces, Thin Films, and Nanostructured Coatings

Creating antibacterial surfaces is the primary approach in preventing the occurrence and diffusion of clinical infections and foodborne diseases as well as in contrasting the propagation of pandemics in everyday life. Proper surface engineering can inhibit microorganism spread and biofilm formation, can contrast antimicrobial resistance (AMR), and can avoid cross-contamination from a contaminated surface to another and eventually to humans. For these reasons, antibacterial surfaces play a key role in many applications, ranging from biomedicine to food and beverage materials, textiles, and objects with frequent human contact. The incorporation of antimicrobial agents within a surface or their addition onto a surface are very effective strategies to achieve this aim and to properly modify many other surface properties at the same time. In this framework, this Special Issue collects research studying several materials and methods related to the antibacterial properties of surfaces for different applications and discussions about the environmental and human-safety aspects

Effect of UHT processing and microconstituent-macromolecule interactions in model liquid systems enriched with insoluble fibre

Very recently, dietary fibre native to wholegrain cereals attracted considerable interest by consumers and researchers alike due to its demonstrated health attributes, such as lowering the blood cholesterol level and reducing the risk of heart disease. Wholegrain oat contains a variety of microconstituents, mainly dietary fibre, proteins, lipids and phytochemicals (phenolic compounds), which can be beneficial to human health. Because of these advantageous properties, food processors incorporate finely milled oat powder into commercial food formulations along with other commonly added ingredients namely as protein, sugar (sucrose) and vegetable oil. However, understanding how ultra-high temperature (UHT) processing and prolonged storage of up to 8 months at ambient temperatures (20 to 25°C) affect these microconstituents in food products is limited. Literature contains insufficient data of molecular interactions that can occur between oat microconstituents and various food ingredients following UHT treatment (120-145°C for very short time) as well as prolonged storage. Therefore, this PhD study aims to unveil the effect of UHT processing and prolonged storage on the bioactivity of oat microconstituents, and to investigate the nature of interactions occurring between these microconstituents and commonly added ingredients in model UHT beverages. This will be achieved by employing numerous analytical and physicochemical techniques, i.e. liquid- and gas-chromatography, Fourier transform infrared (FTIR), fluorescence, UV-vis and circular dichroism spectroscopy, particle size analysis, surface charge analysis, dynamic interfacial tension, molecular docking, and quantum energy calculations. In the first experimental chapter, the influence of UHT treatment (145°C for 8 s) and storage temperatures (22 and 40°C) on phenolic compounds (phenolic acids and avenanthramides), free fatty acids and volatile secondary lipid oxidation products in model oat-beverage formulations containing various concentration of oat powder were examined over 12 weeks. Ferulic acid, followed by p-coumaric acid, was found to be the most abundant phenolic acid in all beverage formulations containing oat. Major avenanthramides (namely A, B and C varities), free fatty acids (palmitic, linoleic and oleic), and volatiles (hexanal and 2-pentyl furan) were also detected from chromatographic analysis. It was reported that following UHT treatment, the total phenolic content (TPC), phenolic acids (particularly p-coumaric and ferulic acids), free fatty acids and avenanthramides decreased. However, prolonged storage of the UHT-treated preparations at ambient temperature had a positive effect on the content of TPC, phenolic acids, avenanthramides, free fatty acids and volatile compounds. In contrast, extended storage at the higher temperature of this study (40°C) decreased TPC and phenolic acid content; the levels of avenanthramides and volatiles increased with time. Overall, UHT treatment and storage conditions had a substantial effect on the composition of bioactive compounds in oat samples of industrial interest. The second experimental chapter examined the molecular interactions and bioactivity of added food ingredients in model oat-based beverages, following UHT processing, at 22°C and for a storage period of twelve weeks. Oat particle concentration of 5% (w/w), sucrose (6.7% w/w), vegetable oil (1.8% w/w) and skim milk powder (2.8% w/w), were utilised to create a number of model beverage formulations with commercial relevance. Results indicate that the insoluble dietary fibre of oat particles contains the majority of phenolic acids, with ferulic acid comprising the largest proportion followed by p-coumaric acid. Application of UHT processing decreased the content of individual phenolic acids (ferulic and p-coumaric acids). In contrast, the level of aforementioned chemical moieties increased with extended storage at 22°C following UHT processing. Among model beverages, those with added milk protein demonstrated a considerable loss of phenolic acids due to the interaction between these micronutrients and milk protein. The nature of molecular interactions was mainly categorised as covalent, with hydrogen bonds playing a supportive role. UHT processing of oat-based beverage formulations facilitated the formation of protein-phenolic acid complexes, which were largely covalent and thermodynamically static in nature. These findings underlined the ability of UHT processing to induce chemical intercations of food ingredients in complex liquid systems. The third experimental chapter examined the significance of UHT processing and storage conditions, at two temperatures (22 and 30°C) for twelve weeks, on the evolution of free fatty acids and lipid oxidation products from oat grains. Work was extended to molecular interactions with other added ingredients. In doing so, model liquid foods of industrial interest were designed utilising finely milled oat particles, skim milk powder, sucrose and vegetable oil. Three major free fatty acids, i.e. palmitic, oleic and linoleic acids were detected across the entire range of preparation. Processing and storage conditions led to the development of major lipid oxidation products i.e. 2-pentyl furan and hexanal. Storage temperature variation from 22 to 30°C had a minor effect on the composition of these microconstituents. However, twelve-week storage exhibited an increase in the level of free fatty acids and secondary oxidation volatiles at both storage temperatures. Addition of milk protein reduced the detected content of free fatty acids and volatiles due to direct interactions among these materials. The molecular nature of the interaction between added milk protein and lipid components of oat grain is of importance for the organoleptic consistency of beverage product concepts. These were examined in some detail to reveal a strong covalent nature in the association between milk protein and free fatty acids. Given the preponderance of covalent interactions between added milk protein in formulations and microconstituents of oat grain, the fourth experimental chapter of this investigation was designed to obtain further insights into their molecular nature. Monodisperse materials were used, i.e. β-casein, which is a major milk protein, and p-coumaric acid at the experimental temperature of 145°C, to identify the specific chemical moiety of the protein responsible for this type of interaction. Analysis focused on spectroscopic fluorescence quenching that provided the Stern-Volmer quenching constant (Ksv), the number of binding sites (n), binding constant (Kb), and the thermodynamic parameters of the free energy of heterotypic association. p-Coumaric acid complexation altered the secondary structure of the protein at the elevated temperature of our treatment by reducing the extent of α-helix and β-sheet conformation thus opening the protein structure to accommodate the ligand in close proximity. Molecular modelling revealed that the probable binding sites of p-coumaric acid are located in the hydrophobic domain of β-casein, where the p-coumaric acid moiety is further linked to the isoleucine27 residue via a hydrogen bonds (2.85 Å). Quantum energy calculations were performed to predict the covalently reacting amino-acid residue with the ligand, and it was concluded that lysine47 can potentially establish a covalent bond (1.50 Å) with p-coumaric acid following UHT processing. This is an entirely unexpected result, since the literature has been arguing mainly for non-covalent interactions between milk proteins and phenolic compounds at neutral pH and ambient temperature

Hydrolases

This book gives a current review of the links between the structure and function of hydrolases and ligases, as well as ideas for better using these critical enzymes. The book is split into two sections: “Cleavage” and “Ligases.” These enzymes are the biggest and most varied family of enzymes, allowing researchers to investigate the structural variety that underpins their different biological roles. In light of recent scientific advances, there is a desire to examine and update our knowledge of these enzymes’ functional and structural changes

Identification and Characterization of Antimicrobial Peptides with Therapeutic Potential

Antimicrobial peptides are key defense molecules adopted by all life forms to prevent infection. They also have other beneficial effects such as boosting immune response, anticancer, and wound healing. The antiviral effects of antimicrobial peptides have laid the foundations for developing new agents to combat seasonal Flu, HIV-1, RSV, Zika, and Ebola. This eBook is constructed to systematically deal with antimicrobial peptides from a variety of natural sources, including fungi, plants, and animals (insects, fish, amphibians, birds, and reptiles). It covers peptide discovery, antimicrobial activity, 3D structure, mechanisms of action and potential applications. Naturally Occurring Antimicrobial Peptides, an eBook published by the journal Pharmaceuticals, provides a helpful introduction to newcomers and refreshes the minds of veterans