Advances in Antimicrobial Peptide Discovery via Machine Learning and Delivery via Nanotechnology

Antimicrobial peptides (AMPs) have been investigated for their potential use as an alternative to antibiotics due to the increased demand for new antimicrobial agents. AMPs, widely found in nature and obtained from microorganisms, have a broad range of antimicrobial protection, allowing them to be applied in the treatment of infections caused by various pathogenic microorganisms. Since these peptides are primarily cationic, they prefer anionic bacterial membranes due to electrostatic interactions. However, the applications of AMPs are currently limited owing to their hemolytic activity, poor bioavailability, degradation from proteolytic enzymes, and high-cost production. To overcome these limitations, nanotechnology has been used to improve AMP bioavailability, permeation across barriers, and/or protection against degradation. In addition, machine learning has been investigated due to its time-saving and cost-effective algorithms to predict AMPs. There are numerous databases available to train machine learning models. In this review, we focus on nanotechnology approaches for AMP delivery and advances in AMP design via machine learning. The AMP sources, classification, structures, antimicrobial mechanisms, their role in diseases, peptide engineering technologies, currently available databases, and machine learning techniques used to predict AMPs with minimal toxicity are discussed in detail

Antimicrobial peptides from the Arctic ascidian Synoicum turgens

The rise in frequency of antibiotic resistant pathogenic bacteria makes the need for new treatment options for previously curable bacterial infections ever more important. In the process of discovering and developing antibacterial agents, one powerful approach has been borrowing wisdom from nature. Antimicrobial peptides (AMPs) are critical components of the innate immune systems found in almost all eukaryotic life forms. Their mode of action includes disruption of the bacterial membrane and to trigger supporting immune responses. Due to these properties, AMPs are considered promising lead structures that can be further developed into commercially available antibiotics to treat or prevent human diseases caused by bacteria. The work conducted in this thesis aims to discover and characterize novel antimicrobial peptides from the Arctic marine ascidian Synoicum turgens by using a marine bioprospecting approach. This includes collection, extraction and fractionation of biomass, antibacterial bioactivity testing and AMP isolation followed by chemical and biological characterization. For one isolated peptide class, truncated versions were prepared, aiming to produce shorter, linear variants with retained antimicrobial activity. In paper I, turgencin A and B and their oxidized derivatives were discovered through bioassay-guided purification. These peptides had an unusual disulfide connectivity, rarely seen in marine AMPs. Both turgencin A and B were potently active against all assayed bacterial strains. Membrane assays showed that the peptides cause bacterial membrane disruption within a few seconds. Turgencin A and B also displayed some cytotoxic activity against two human cell lines. Oxidation of the methionine present in both peptides decreased the bioactivities in all assays. Turgencin A, being the most potent AMP, was subject for sequence analysis and prediction of antimicrobial potential of different regions in paper II. Truncated and modified variants of turgencin A were synthetically produced to make smaller AMPs with the potential of being developed into antimicrobial drug leads. These 10-mer peptides, named StAMP-1–11, were made using an amino acid replacement strategy. Some of the Trp enriched peptides had similar bactericidal activity as the parent peptide turgencin A, and no cytotoxic activity against the mammalian cell lines. During turgencin isolation, a series of other smaller peptides were discovered in the same extracts presumably with antimicrobial activity. These isolated and characterized ~2 kDa, cysteine-rich peptides (CRPs) (described in paper III), were named St-CRP-1 and St-CRP-2 and contained 18-19 amino acids. The St-CRPs shared a disulfide connectivity pattern with alpha-defensins, had a neutral net charge, moderate antibacterial activity and showed no cytotoxicity. In addition, the introduction section provides background information on topics related to the thematic of the articles. This includes an introduction to bacteria, antibiotics and antibiotic resistance, AMPs, ascidians, and the marine environment

Protein Structure

Since the dawn of recorded history, and probably even before, men and women have been grasping at the mechanisms by which they themselves exist. Only relatively recently, did this grasp yield anything of substance, and only within the last several decades did the proteins play a pivotal role in this existence. In this expose on the topic of protein structure some of the current issues in this scientific field are discussed. The aim is that a non-expert can gain some appreciation for the intricacies involved, and in the current state of affairs. The expert meanwhile, we hope, can gain a deeper understanding of the topic

Discovery of botanical antimicrobial peptides via mass spectrometric approaches: An exploration of Amaranthus spp.

(Under the direction of Leslie M. Hicks)The emergence of antimicrobial resistant microbes has generated an urgent need for novel antibiotic compounds to maintain modern health care and agricultural practices. Botanical natural products, such as antimicrobial peptides (AMPs), can contribute to a multimodal solution to combat multi-drug resistant pathogens. This dissertation explores mass spectrometric methods for botanical AMP discovery through the investigation two species from the genus Amaranthus emphasizing complementary activity- and prediction-based approaches. First, a novel proline-rich antibacterial AMP, Atr-AMP1, is discovered via PepSAVI-MS from an Amaranthus tricolor extract (Chapter 2). The unusual sequence of Atr-AMP1 combined with a heterogenous population of peptidoforms resulted in de novo sequencing challenges. This case study highlighted the barrier to sequence characterization during peptide discovery in plant species lacking high quality genomic resources. As a result, mass spectrometric methods which provide information on amino acid/post-translation modification were compiled to assist future sequencing efforts (Chapter 3).The A. tricolor peptide library is further examined for in silico predicted AMPs which were not identified via activity-based PepSAVI-MS (Chapter 4). Bottom-up proteomic analysis provided evidence for the translation of seven putative AMPs including Atr-LTP1, a novel lipid transfer protein, with validated activity against Klebsiella pneumoniae. A. tricolor AMPs were further investigated via a truncated synthetic analog of predicted defensin Atr-DEF2 revealing the role of outer membrane permeabilization and transition metal sequestration in observed activity against the gram negative pathogen E. coli (Chapter 5). Then, proteomic/peptidomics guided by in silico AMP predictions were used to confirm the translation of 13 predicted AMPs in the seeds of the alternative grain A. hypochondriacus (Chapter 6). An in vitro gastrointestinal digestion confirmed that at least six of the AMPs are resistant to proteolysis and may survive human consumption of A. hypochondriacus derived foodstuffs. Together, the work compiled in this dissertation emphasizes that no single discovery strategy is sufficient to capture the full suite of AMPs expressed by a plant species. Instead, pairing bioactivity- and predictive-based approaches facilitates identification of AMPs of known families while activity screening can be advantageous to discovery of novel peptides families. Integrating these two paradigms is critical for comprehensive AMP discovery.Doctor of Philosoph

In-silico optimization and molecular validation of putative anti-HIV antimicrobial peptides for therapeutic purpose

Philosophiae Doctor - PhDAIDS is considered a pandemic causing millions of deaths worldwide and a cure for this disease is still not available. Failure to implement early treatments due to the poor diagnostic methods and ineffective therapeutic regimens to treat HIV patients to achieve complete viral eradication from the human body has encouraged the escalation of this disease at an exponential rate. Though the current treatment regimens (High Active Antiretroviral Therapy) have aided in increasing the lifespan of HIV patients, it still suffers from some shortcomings such as adverse side effects and non-eradication of the virus. Thus, there is a need for a non-toxic therapeutic regimen to stop further infection of HIV-infected patients. Antimicrobial Peptides (AMPs) are naturally occurring peptides which are components of the first line of defence of many organisms against infections and have been proven to be promising therapeutic agents against HIV. The use of AMPs as anti-microbial agents is due to the fact that most AMPs have a net positive charge and are mostly hydrophobic molecules. These features allow AMPs to be site directed electro-statistically to the mostly negatively charged pathogens. In a previous study, a number of novel anti-HIV AMPs was identified using a predictive algorithm Profile Hidden Markov Models (HMMER). The AMP's threedimensional structures were predicted using an in-silico modelling tool I-TASSER and an insilico protein-peptide interaction study of the AMPs to HIV protein gp120 was performed using PatchDock. Five AMPs were identified to bind gp120, at the site where gp120 interacts with CD4 to prevent HIV invasion and HIV replication. Therefore, the aims of this research were to perform in-silico site-directed mutation on the parental anti-HIV AMPs to increase their binding affinity to the gp120 protein, validate the anti-HIV activity of these peptides and confirm the exclusivity of this activity by testing possible anti-bacterial and anti-cancer activities of the AMPs. Firstly, the five parental anti-HIV AMPs were used to generate mutated AMPs through insilico site-directed mutagenesis. The AMPs 3-D structures were determined using I-TASSER and the modelled AMPs were docked against the HIV protein gp120 using PatchDock. Secondly, an "in house" Lateral Flow Device (LFD) tool developed by our industrial partner, Medical Diagnostech (Pty) Ltd, was utilised to confirm the in-silico docking results. Furthermore, the ability of these AMPs to inhibit HIV-1 replication was demonstrated and additional biological activities of the peptides were shown on bacteria and cancer cell lines. In an effort to identify AMPs with increased binding affinity, the in-silico results showed that two mutated AMPs Molecule 1.1 and Molecule 8.1 bind gp120 with high affinity, at the point where gp120 bind with CD4. The molecular binding however showed that only Molecule 3 and Molecule 7 could prevent the interaction of gp120 protein and CD4 surface protein of human cells, in a competitive binding assay. Additionally, the testing of the anti-HIV activity of the AMPs showed that Molecule 7, Molecule 8 and Molecule 8.1 could inhibit HIV-1 NL4-3 with maximal effective concentration (EC₅₀) values of 37.5 μg/ml and 93.75 μg/ml respectively. The EC₅₀ of Molecule 8.1 was determined to be around 12.5 μg/ml. This result looks promising since 150 μg/ml of the AMPs could not achieve 80% toxicity of the human T cells, thus high Therapeutics Index (TI) might be obtained if 50% cytotoxic concentration (CC₅₀) is established. Further biological activity demonstrates that Molecule 3 and Molecule 7 inhibited P. aeruginosa completely after 24 hours treatment with peptide concentrations ranging from 0.5 mg/ml to 0.03125 mg/ml. Nevertheless, moderate inhibition was observed when CHO, HeLa, MCF-7 and HT-29 were treated with these peptides at peptides concentration of 100 μg/ml. The ability of these AMPs to block the entrance of HIV via the binding to CD4 of the host cells is a good concept since they pave the way for the design of anti-HIV peptide-based drugs Entry Inhibitors (FIs) or can be exploited in the production microbicide gels/films to suppress the propagation of the virus.DST-NIC/Minte

Exploring the chemical space of post-translationally modified peptides in Streptomyces with machine learning

The ongoing increase in antimicrobial resistance combined with the low discovery of novel antibiotics is a serious threat to our health care. Genome mining has given new potential to the field of natural product discovery, as thousands of biosynthetic gene clusters (BGCs) are discovered for which the natural product is not known.Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a highly diverse class of natural products. The large number of different modifications that can be applied to a RiPP results in a large variety of chemical structures, but also stems from a large genetic variety in BGCs. As a result, no single method can effectively mine for all RiPP BGCs, making it an interesting source for new molecules.In this thesis, new methods are explored to mine genomes for the BGCs of novel RiPP variants, with a focus on discovering RiPPs that have new modifications. RRE-Finder is a new tool for the detection of RiPP Recognition Elements, domains that are often found in RiPP BGCs. DecRiPPter is another tool that employs machine learning models to discover new RiPP precursor genes encoded in the genomes. Both tools can be used to prioritize novel RiPP BGCs. Two candidate BGCs are characterized, one of which could be shown to specify a new RiPP, validating the approach.Grant 731.014.206 (Syngenopep, TKI Chemie) from the Dutch Research Council (NWO)Microbial Biotechnolog

Modelling the genomic structure, and antiviral susceptibility of Human Cytomegalovirus

Human Cytomegalovirus (HCMV) is found ubiquitously in humans worldwide, and once acquired, the infection persists within the host throughout their life. Although Immunocompetent people rarely are affected by HCMV infections, their related diseases pose a major health problem worldwide for those with compromised or suppressed immune systems such as transplant recipients. Additionally, congenital transmission of HCMV is the most common infectious cause of birth defects globally and is associated with a substantial economic burden. This thesis explores the application of statistical modelling and genomics to unpick three key areas of interest in HCMV research. First, a comparative genomics analysis of global HCMV strains was undertaken to delineate the molecular population structure of this highly variable virus. By including in-house sequenced viruses of African origin and by developing a statistical framework to deconvolute highly variable regions of the genome, novel and important insights into the co-evolution of HCMV with its host were uncovered. Second, a rich database relating mutations to drug sensitivity was curated for all the antiviral treated herpesviruses. This structured information along with the development of a mutation annotation pipeline, allowed the further development of statistical models that predict the phenotype of a virus from its sequence. The predictive power of these models was validated for HSV1 by using external unseen mutation data provided in collaboration with the UK Health Security Agency. Finally, a nonlinear mixed effects model, expanded to account for Ganciclovir pharmacokinetics and pharmacodynamics, was developed by making use of rich temporal HCMV viral load data. This model allowed the estimation of the impact of immune-clearance versus antiviral inhibition in controlling HCMV lytic replication in already established infections post-haematopoietic stem cell transplant

Atividades antibacteriana e antibiofilme de dois peptídeos helicoidais : PA-MAP 1.9 e ECDBS1R5

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2019.Os peptídeos antimicrobianos (PAMs) vêm sendo amplamente investigados, representando uma estratégia promissora no combate a células planctônicas e biofilmes bacterianos multirresistentes. Assim, o presente trabalho teve como objetivo a caracterização funcional e estrutural de dois PAMs helicoidais com potencial antibacteriano, antibiofilme e antiinfeccioso, denominados Pa-MAP 1.9 e EcDBS1R5. Inicialmente, foi observado que ambos os peptídeos de estudo apresentam atividades contra cepas Gram-negativas e Gram-positivas, susceptíveis e resistentes, sem causar hemólise ou comprometer a viabilidade de linhagens celulares saudáveis proveniente de mamíferos. Em adição, o peptídeo Pa-MAP 1.9 demonstrou atividade deletéria contra biofilmes a baixas concentrações (1,1-3,0 μM), resultando não somente na inibição da formação de biofilmes, como também na erradicação de biofilmes pré-formados por E. coli e Klebsiella pneumoniae KpC+. A atividade antibiofilme para EcDBS1R5 foi também confirmada, uma vez que este peptídeo reduziu o volume e altura de biofilmes de P. aeruginosa pré-formados, comprometendo também a viabilidade de células bacterianas nos biofilmes. Ademais, o potencial anti-infeccioso de EcDBS1R5 foi avaliado, resultando em uma redução de 100 vezes na carga de P. aeruginosa em infecções cutâneas. Ensaios in vivo com o peptídeo Pa-MAP 1.9 ainda estão em andamento. Em contrapartida, o mecanismo de ação de Pa-MAP 1.9 frente a cepas de E. coli foi aqui investigado, sugerindo sua atuação em alvos intracelulares. Além disso, estudos biofísicos mostram que Pa-MAP 1.9 foi mais eficiente na interação com vesículas e membranas aniônicas (bactérias Gram-negativas), em comparação àquelas enriquecidas com colesterol (células de mamíferos). Estruturalmente, ambos os peptídeos foram submetidos a experimentos de dicroísmo circular (DC), revelando a presença de altos conteúdos de α-hélice em ambientes hidrofóbicos e aniônicos, contrário à conformações em random coil observadas em ambientes aquosos. As estruturas tridimensionais desses peptídeos foram detalhadamente estudadas e corroboram os dados de DC, reforçando a preferência conformacional em α- hélice anfipática com regiões terminais flexíveis tanto para Pa-MAP 1.9 quanto para EcDBS1R5 em ambientes que mimetizam membranas bacterianas. Dessa forma, os PAMs helicoidais aqui estudados se mostram promissores candidatos no desenvolvimento de novas ferramentas biotecnológicas contra infecções causadas por células platônicas e biofilmes bacterianos.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Apoio à Pesquisa do Distrito Federal (FAP/DF).Antimicrobial peptides (AMPs) have been largely investigated, representing a promising strategy in the combat of multidrug resistant planktonic bacteria and biofilms. Thus, the present study focused on the functional and structural characterization of two helical AMPs with antibacterial, antibiofilm and anti-infective potential, denominated Pa-MAP 1.9 and EcDBS1R5. Initially, it was observed that both peptides present activities against Gramnegative and Gram-positive, susceptible and resistant strains, without causing hemolysis or compromising healthy mammalian cell lines viability. Moreover, the peptide Pa-MAP 1.9 demonstrated deleterious activities against biofilms at low concentrations (1.1-3.0 μM), resulting not only in the inhibition of biofilm formation, but also in the eradication of E. coli and K. pneumoniae KpC+ pre-formed biofilms. The antibiofilm activity for EcDBS1R5 was also confirmed, as this peptide decreased both the volume and height of pre-formed P. aeruginosa biofilms, also compromising the viability of bacterial cells within biofilms. Moreover, the anti-infective potential of EcDBS1R5 was evaluated, reducing 100-times P. aeruginosa counts in cutaneous infections. In vivo assays for Pa-MAP 1.9 are in progress. In contrast, the mechanism of action of Pa-MAP 1.9 toward E. coli was here investigated, suggesting its action on intracellular targets. Moreover, biophysical studies showed that Pa- MAP 1.9 was more efficient in interacting with anionic vesicles and membranes (Gramnegative bacteria), in comparison to those enriched with cholesterol (mammalian cells). Structurally, both peptides were submitted to circular dichroism (CD) experiments, revealing the presence of high α-helical contents in hydrophobic and anionic environments, in contrary to random coil conformations observed in hydrophilic environment. The tridimensional structures of these peptides were studied in detail and corroborate the CD data, reinforcing the conformational preference in amphipathic α-helix with flexible termini for both Pa-MAP 1.9 and EcDBS1R5 in environments that mimic bacterial membranes. Thus, the helical AMPs here studied appear as promising candidates in the development of biotechnological tools against infections caused by bacterial planktonic cells and biofilms