Analogs of the Frog-skin Antimicrobial Peptide Temporin 1Tb Exhibit a Wider Spectrum of Activity and a Stronger Antibiofilm Potential as Compared to the Parental Peptide

The frog skin-derived peptide Temporin 1Tb (TB) has gained increasing attention as novel antimicrobial agent for the treatment of antibiotic-resistant and/or biofilm-mediated infections. Nevertheless, such a peptide possesses a preferential spectrum of action against Gram-positive bacteria. In order to improve the therapeutic potential of TB, the present study evaluated the antibacterial and antibiofilm activities of two TB analogs against medically relevant bacterial species. Of the two analogs, TB_KKG6A has been previously described in the literature, while TB_L1FK is a new analog designed by us through statistical-based computational strategies. Both TB analogs displayed a faster and stronger bactericidal activity than the parental peptide, especially against Gram-negative bacteria in planktonic form. Differently from the parental peptide, TB_KKG6A and TB_L1FK were able to inhibit the formation of Staphylococcus aureus biofilms by more than 50% at 12 μM, while only TB_KKG6A prevented the formation of Pseudomonas aeruginosa biofilms at 24 μM. A marked antibiofilm activity against preformed biofilms of both bacterial species was observed for the two TB analog when used in combination with EDTA. Analysis of synergism at the cellular level suggested that the antibiofilm activity exerted by the peptide-EDTA combinations against mature biofilms might be due mainly to a disaggregating effect on the extracellular matrix in the case of S. aureus, and to a direct activity on biofilm-embedded cells in the case of P. aeruginosa. Both analogs displayed a low hemolytic effect at the active concentrations and, overall, TB_L1FK resulted less cytotoxic toward mammalian cells. Collectively, the results obtained demonstrated that subtle changes in the primary sequence of TB may provide TB analogs that, used alone or in combination with adjuvant molecules such as EDTA, exhibit promising features against both planktonic and biofilm cells of medically relevant bacteria

Prediction of Drug Loading in the Gelatin Matrix Using Computational Methods

The delivery of drugs is a topic of intense research activity in both academia and industry with potential for positive economic, health, and societal impacts. The selection of the appropriate formulation (carrier and drug) with optimal delivery is a challenge investigated by researchers in academia and industry, in which millions of dollars are invested annually. Experiments involving different carriers and determination of their capacity for drug loading are very time-consuming and therefore expensive; consequently, approaches that employ computational/theoretical chemistry to speed have the potential to make hugely beneficial economic, environmental, and health impacts through savings in costs associated with chemicals (and their safe disposal) and time. Here, we report the use of computational tools (data mining of the available literature, principal component analysis, hierarchical clustering analysis, partial least squares regression, autocovariance calculations, molecular dynamics simulations, and molecular docking) to successfully predict drug loading into model drug delivery systems (gelatin nanospheres). We believe that this methodology has the potential to lead to significant change in drug formulation studies across the world

Antimicrobial peptides and their interaction with biofilms of medically relevant bacteria

Biofilm-associated infections represent one of the major threats of the modern medicine. Biofilmforming bacteria are encased in a complex mixture of extracellular polymeric substances (EPS) and acquire properties that render them highly tolerant to conventional antibiotics and host immune response. Therefore, there is a pressing demand of new drugs active against microbial biofilms. In this regard, antimicrobial peptides (AMPs) represent an option taken increasingly in consideration. After dissecting the peculiar biofilm features that may greatly affect the development of new antibiofilm drugs, the present article provides a general overview of the rationale behind the use of AMPs against biofilms of medically relevant bacteria and on the possible mechanisms of AMP antibiofilm activity. An analysis of the interactions of AMPs with biofilm components, especially those constituting the EPS, and the obstacles and/or opportunities that may arise from such interactions in the development of new AMP-based antibiofilm strategies is also presented and discussed

Biophysical mechanisms of membrane perturbation and signal transduction produced by proteins and peptides

My primary research interest is focused on the field of cellular electrical activity, ranging from the ion channels that generates it, up to the study of intracellular processes regulating it, and new generation of drugs. For this purpose, during my Ph.D. I have learnt and improved different cutting-edge techniques, i.e. the patch-clamp technique, the fluorescence imaging, and the synthesis and use of model membranes. Moreover, to explore particular aspects of these molecular mechanisms and to overcome the issues raised during the investigations, non-conventional strategies were employed, even requiring the development of specific devices not commercially available. In summary, my Ph.D. thesis is focused on two projects: the biophysical characterization of a particular class of membrane active peptides, and the modulation of visual phototransduction in vertebrate cones. In the first project, I investigated the mechanism of membrane perturbation of cell-penetrating and antimicrobial peptides using the patch-clamp technique. Cell-penetrating peptides (CPPs) are short peptides that are able to cross the cell membrane via energy-dependent and/or independent mechanisms, with low toxicity and without the use of specific receptors. This ability is preserved even when CPPs are conjugated with a large cargo, thus representing an innovative pharmacological tool for the diffusion of large and hydrophilic drugs into the cells. Despite the mechanism of cellular uptake is still debated in literature, it has been proved that it can occur by either direct translocation or endocytosis. In the latter case, though, the cargo-peptide complex often remains trapped inside the endocytic vesicles and is not able to reach its therapeutic target. A possible solution to this problem could be found in another class of small peptides, similar to CPPs, i.e. the antimicrobial peptides (AMPs). AMPs are 12-50 amino acids long peptides, which represent an essential part in the innate immune system in most organisms. Indeed, they are among the first defensive molecules released during infections and their activity is direct thorough the membrane of bacteria, causing its destruction and consequently the death of the pathogen. Therefore, the ability of AMPs to disrupt biological membranes could be exploited to improve the CPPs escape from the endocytic vesicles in addition to, of course, their application as a novel class of antibiotics. The idea is to conjugate the CPP with a molecule that possess an antimicrobial activity, which can destroy the vesicle membrane, and help the complex to reach its target once it has been internalized in the cell. On this ground, the first project I carried out regards the study of a novel chimeric peptide, CM18-Tat11, composed of the antimicrobial peptide CM18 (a cecropin-mellitin hybrid peptide) linked to the cell-penetrating peptide Tat11 (derived from the basic domain of HIV-1 Tat protein). In particular, I investigated the membrane perturbing activity of this peptide (and of its elements) using the patch-clamp technique and operating under strictly physiological conditions. This study has been carried out by recording the ion current flowing through the channels formed by these peptides (if any), once inserted in the membrane of Chinese hamster ovary (CHO) cells. In these experiments, the peptides were applied to (and removed from) the extracellular CHO membrane in ~50 ms with a computer-controlled microperfusion system. Therefore, besides assessing ion channel characteristics, the dynamics of pore formation and disaggregation could be precisely evaluated as well. I found that CM18-Tat11 produces a large and irreversible plasma membrane lysis, at concentration where CM18 and Tat11 give instead a nearly reversible membrane permeabilization and no perturbation, respectively. Furthermore, using the same method, I studied the biophysical characteristic of another antimicrobial peptide, called CM12, which sequence was obtained from the optimization of CM18. When applied on CHO, CM12 and CM18 produce voltage-independent membrane permeabilization, and no single-channel events were detected at low peptides concentration. These results indicate that both peptides form pores according to a toroidal model, in which the lipid layer bends continuously through the pore so that the core is formed by both lipid head groups and the peptides. Finally, I have studied the single-channels properties generated by the pore-forming peptide alamethicin (Alm) F50/5 and its [L-Glu(OMe)7,18,19] analog inserted in a natural membrane and in giant unilamellar vesicles (GUVs). The possibility to compare the channel activity in the precisely controlled lipid environment of GUVs, with the one recorded in a natural membrane, will open new possibilities in the biophysical characterization of the pores. The second project of this thesis is focused on the study of the physiological role of the calcium sensor GCAP3 (guanylate cyclase activated protein 3) in the phototransduction cascade in zebrafish. I pursued this study simulating the over expressions and the knockdown of this protein, through the delivery of zGCAP3, or of its monoclonal antibody, into zebrafish cone cytoplasm, while recording their photorensponses with the patch-clamp technique. The proteins were administered inside the cone via the patch pipette thanks to an intracellular perfusion system developed in this thesis. This system allows the delivery of exogenous molecules inside the cell with a controlled timing, by expelling them with a small teflon tube inserted into the pipette lumen controlled by a microperfusion apparatus. Results indicated that the increase in the concentration in zGCAP3 did not altered significantly the light response, while the perfusion with the antibody anti-zGCAP3 caused the progressive fall of the dark current, together with the progressive slowing down of the flash response kinetics. The surprising lack of an effect of zGCAP3 incorporation, suggests that the endogenous number of zGCAP3 is saturating, therefore any further increase of this sensor is ineffective. However, the effects of the antibody can be explained as an inhibition of the target enzyme of zGCAP3, which is the guanylate cyclase (GC). Finally, no experiments mentioned above would have been accomplished without the development of a “pressure-polishing” system, which makes it possible to modify the geometry of the patch-clamp pipette. The pipette shank (the final part of the pipette) is, in fact, very long and tapered, thus generating a high resistance to the passage of ions and molecules, and making very difficult to perfuse efficiently the cell with the internal perfusion. The pressure polishing setup I developed enlarged the patch pipette shank, using a calibrated combination of heat and air pressure. These pipettes minimized errors in membrane potential control and allowed the insertion of teflon tubes in the pipette lumen very close to its tip

Reassessing the Host Defense Peptide Landscape

Current research has demonstrated that small cationic amphipathic peptides have strong potential not only as antimicrobials, but also as antibiofilm agents, immune modulators, and anti-inflammatories. Although traditionally termed antimicrobial peptides (AMPs) these additional roles have prompted a shift in terminology to use the broader term host defense peptides (HDPs) to capture the multi-functional nature of these molecules. In this review, we critically examined the role of AMPs and HDPs in infectious diseases and inflammation. It is generally accepted that HDPs are multi-faceted mediators of a wide range of biological processes, with individual activities dependent on their polypeptide sequence. In this context, we explore the concept of chemical space as it applies to HDPs and hypothesize that the various functions and activities of this class of molecule exist on independent but overlapping activity landscapes. Finally, we outline several emerging functions and roles of HDPs and highlight how an improved understanding of these processes can potentially be leveraged to more fully realize the therapeutic promise of HDPs

Cheminformatics Tools to Explore the Chemical Space of Peptides and Natural Products

Cheminformatics facilitates the analysis, storage, and collection of large quantities of chemical data, such as molecular structures and molecules' properties and biological activity, and it has revolutionized medicinal chemistry for small molecules. However, its application to larger molecules is still underrepresented. This thesis work attempts to fill this gap and extend the cheminformatics approach towards large molecules and peptides. This thesis is divided into two parts. The first part presents the implementation and application of two new molecular descriptors: macromolecule extended atom pair fingerprint (MXFP) and MinHashed atom pair fingerprint of radius 2 (MAP4). MXFP is an atom pair fingerprint suitable for large molecules, and here, it is used to explore the chemical space of non-Lipinski molecules within the widely used PubChem and ChEMBL databases. MAP4 is a MinHashed hybrid of substructure and atom pair fingerprints suitable for encoding small and large molecules. MAP4 is first benchmarked against commonly used atom pairs and substructure fingerprints, and then it is used to investigate the chemical space of microbial and plants natural products with the aid of machine learning and chemical space mapping. The second part of the thesis focuses on peptides, and it is introduced by a review chapter on approaches to discover novel peptide structures and describing the known peptide chemical space. Then, a genetic algorithm that uses MXFP in its fitness function is described and challenged to generate peptide analogs of peptidic or non-peptidic queries. Finally, supervised and unsupervised machine learning is used to generate novel antimicrobial and non-hemolytic peptide sequences

Antimicrobial Peptides Design by Evolutionary Multiobjective Optimization

<div><p>Antimicrobial peptides (AMPs) are an abundant and wide class of molecules produced by many tissues and cell types in a variety of mammals, plant and animal species. Linear alpha-helical antimicrobial peptides are among the most widespread membrane-disruptive AMPs in nature, representing a particularly successful structural arrangement in innate defense. Recently, AMPs have received increasing attention as potential therapeutic agents, owing to their broad activity spectrum and their reduced tendency to induce resistance. The introduction of non-natural amino acids will be a key requisite in order to contrast host resistance and increase compound's life. In this work, the possibility to design novel AMP sequences with non-natural amino acids was achieved through a flexible computational approach, based on chemophysical profiles of peptide sequences. Quantitative structure-activity relationship (QSAR) descriptors were employed to code each peptide and train two statistical models in order to account for structural and functional properties of alpha-helical amphipathic AMPs. These models were then used as fitness functions for a multi-objective evolutional algorithm, together with a set of constraints for the design of a series of candidate AMPs. Two ab-initio natural peptides were synthesized and experimentally validated for antimicrobial activity, together with a series of control peptides. Furthermore, a well-known Cecropin-Mellitin alpha helical antimicrobial hybrid (CM18) was optimized by shortening its amino acid sequence while maintaining its activity and a peptide with non-natural amino acids was designed and tested, demonstrating the higher activity achievable with artificial residues.</p></div