Polymers for Delivering a Substance into a Cell

Disclosed herein are polymers that can be made cationic and used to deliver a substance into a cell. Also disclosed are pharmaceutical compositions comprising the polymers and methods of using the polymers

A Focused Poly(Aminoether) Library for Transgene Delivery to Cancer Cells

abstract: Cancer diseases are among the leading cause of death in the United States. Advanced cancer diseases are characterized by genetic defects resulting in uncontrollable cell growth. Currently, chemotherapeutics are one of the mainstream treatments administered to cancer patients but are less effective if administered in the later stages of metastasis, and can result in unwanted side effects and broad toxicities. Therefore, current efforts have explored gene therapy as an alternative strategy to correct the genetic defects associated with cancer diseases, by administering genes which encode for proteins that result in cell death. While the use of viral vectors shows high level expression of the delivered transgene, the potential for insertion mutagenesis and activation of immune responses raise concern in clinical applications. Non-viral vectors, including cationic lipids and polymers, have been explored as potentially safer alternatives to viral delivery systems. These systems are advantageous for transgene delivery due to ease of synthesis, scale up, versatility, and in some cases due to their biodegradability and biocompatibility. However, low efficacies for transgene expression and high cytotoxicities limit the practical use of these polymers. In this work, a small library of twenty-one cationic polymers was synthesized following a ring opening polymerization of diglycidyl ethers (epoxides) by polyamines. The polymers were screened in parallel and transfection efficacies of individual polymers were compared to those of polyethylenimine (PEI), a current standard for polymer-mediated transgene delivery. Seven lead polymers that demonstrated higher transgene expression efficacies than PEI in pancreatic and prostate cancer cells lines were identified from the screening. A second related effort involved the generation of polymer-antibody conjugates in order to facilitate targeting of delivered plasmid DNA selectively to cancer cells. Future work with the novel lead polymers and polymer-antibody conjugates developed in this research will involve an investigation into the delivery of transgenes encoding for apoptosis-inducing proteins both in vitro and in vivo.Dissertation/ThesisM.S. Chemical Engineering 201

Current Knowledge and Future Directions in Developing Strategies to Combat Pseudomonas aeruginosa Infection.

In the face of growing antimicrobial resistance, there is an urgent need for the development of effective strategies to target Pseudomonas aeruginosa. This metabolically versatile bacterium can cause a wide range of severe opportunistic infections in patients with serious underlying medical conditions, such as those with burns, surgical wounds or people with cystic fibrosis. Many of the key adaptations that arise in this organism during infection are centered on core metabolism and virulence factor synthesis. Interfering with these processes may provide a new strategy to combat infection which could be combined with conventional antibiotics. This review will provide an overview of the most recent work that has advanced our understanding of P. aeruginosa infection. Strategies that exploit this recent knowledge to combat infection will be highlighted alongside potential alternative therapeutic options and their limitations.Herchel Smith Postdoctoral Fellowshi

IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD

Structure-based design of inhibitors of CXCR4.

Metastasis is a complex process requiring directed migration of metastatic cells to favorable microenvironments. Increased CXCR4 expression has been implicated in more invasive, aggressive and metastatic tumor phenotypes and poor patient survival in twenty-three forms of cancer. CXCR4 has been linked to cancer metastasis and CXCR4 expression on the cell surface of tumor cells has been linked to increased migration and homing of neoplastic cells to sites where stromal cells express the chemokine CXCL 12 such as the lung and bone marrow. In this dissertation, we will utilize structure based drug design to identify inhibitors of CXCR4 targeting the extracellular surface of the receptor, as well as the intracellular interface between the GPCR and G-protein. Our screens of the extracellular surface identified one compound, ECLVS14, which inhibits chemotaxis with an IC50 value of 5 j..IM, and is highly selective for CXCR4 without significant cytotoxicity. Subsequent QSAR analysis of the structure of this inhibitor reveals the importance of the 1-[bis (phenyl methyl) amino] methyl moiety and the fact that electronegative modifications of the terminal benzene enhance activity. Subsequent Molecular dynamics simulations of the compound in complex with CXCR4 reveal that the compound induces significant modifications of the receptor structure. Our intracellular screens represent a novel screening strategy targeting the intracellular region of CXGR4 interacting with Gai, which identified ten compounds selectively inhibiting GXGR4 with IG50 values of 10 IJM or less. Three of the most active compounds from the extracellular and intracellular screens were tested in an in vivo anti-metastatic animal model, successfully demonstrating the anti-metastatic activity of these compounds. In total this work demonstrates that structure based drug design utilizing in silico analysis in combination with in vitro and in vivo testing can be utilized to develop novel lead compounds which can function as anti-metastatics

Evolution and Prevention of Antibiotic Resistance: Small Molecule Inhibitors of Bacterial Recombination Enzymes

Antibiotic resistant bacteria are an ever-increasing problem for the modern chemotherapy of bacterial infectious diseases. The loss of effective antibiotic therapies due to antibiotic resistance and the withering antibiotic pipeline are resulting in a reemergence in deaths from bacterial infections. New strategies are needed to combat pathogenic bacteria and in this context bacterial targets involved in the development of resistance are emerging an intriguing candidates for inhibition studies. Recent evidence suggests that bacterial stress response pathways (i.e., SOS and competence for transformation) are responsible for accelerated genetic changes that ultimately establish antibiotic resistance. Intervening in these pathways by small molecule inhibition of key recombination enzymes, RecA and EndA, would impact the DNA repair, SOS mutagenesis and recombination-based horizontal gene transfer activities of these enzymes and hinder the acquisition of antibiotic resistance. Bacteria having loss-of-function mutations in the recA gene are more sensitive to antibiotic treatment and develop resistance more slowly or not at all. In addition, endA-null strains of S. pneumoniae have diminished transformation efficiencies and are unable to acquire resistance-conferring DNA. Therefore, we believe chemotherapeutic agents that impart these bacterial phenotypes could act synergistically with currently prescribed antibiotics to prevent the accumulation of populations that are resistant to them. Towards this goal, we sought to identify properly designed inhibitors of RecA and EndA. High-throughput screening (HTS) is recognized as a powerful tool in drug discovery to identify target-specific lead compounds. We developed rational high-throughput screening programs to discover small-molecule inhibitors of RecA and EndA. Through these studies, we have identified novel chemical classes that specifically target RecA or EndA and demonstrate that these enzymes hold potential as novel targets in the treatment of bacterial infections.Doctor of Philosoph

Microsphere based protease assays and high throughput screening of bacterial toxin proteases

Proteases, proteins which cleave peptide bonds in other proteins, are a large and varied group of proteins which regulate a variety of physiological processes. Methodologies to study proteases are often protease specific and often differ greatly from the roles proteases play in vivo. In vitro protease assays often use peptide based substrates, which do not take into account highly specific interactions distal from the proteolytic site of peptide cleavage on protease substrates. In the work described here we have developed a microsphere based protease assay, capable of using full length protease substrates, and have successfully measured proteolytic activity via loss of fluorescence as measured by flow cytometry. This assay is capable of being used in high throughput screening for small molecule inhibitors for proteases of medical relevance. Screening of chemical libraries against the Bacillus anthracis Lethal factor metalloprotease and the Clostridium botulinum Neurotoxin type A Light Chain metalloprotease has led to the discovery of small molecule inhibitors for both of these pathogenic proteases. The compound ebselen has been shown to inhibit Botulinum Neurotoxin type A Light Chain with an IC50 value in the low μM range. Additional small molecule inhibitors for Botulinum neurotoxin type A Light Chain as well as for anthrax lethal factor have also been discovered by this methodology. This work shows the potential for microsphere based protease assays in discovery of small molecule protease inhibitors and can be adapted to any protease/substrate system of interest in a multiplex setup. Additional work with these proteases has also led to the discovery of novel solution based kinetics models and shows promise to validate microsphere based protease kinetics using the same system

Leishmaniasis

Leishmaniasis is a major global health challenge, affecting approximately 12 million of the poorest people in 100 countries. It is a deforming and fatal disease in the visceral form. Therapies for leishmaniasis are numerically restricted, basically consisting of the administration of miltefosine, pentavalent antimonials, amphotericin B, or pentamidine. This is an important vulnerability against therapy efficiency that must be overcome by the scientific community. This book discusses important aspects of the disease, such as treatment, epidemiology, and molecular and cell biology. The information contained herein is important for young researchers as they seek to develop safe and effective treatments for this neglected tropical disease

Current and emerging therapies for corneal infection: a clinical and laboratory study

Corneal infection or infectious keratitis (IK) is a major cause for corneal blindness worldwide. Broad-spectrum antimicrobial therapy is currently the mainstay of treatment for IK, but the efficacy is being challenged by the emergence of antimicrobial resistance. Host defense peptides (HDPs), also known as antimicrobial peptides (AMPs), are evolutionarily conserved molecules of innate immune system that are found in all kingdoms of life. HDPs have shown promise as a novel class of antimicrobial therapeutics due to their broad-spectrum and rapid antimicrobial activity against a wide array of infection with minimal risk of developing resistance. At the ocular surface, HDPs, particularly human cathelicidin (LL-37) and human beta-defensins (HBDs), have been shown to play a vital role during IK. The first part of this work (Chapter 2 to Chapter 4) consisted of a body of work examining the epidemiology, causes, clinical characteristics, outcomes, and prognostic factors of IK in Nottingham, UK. IK was shown to be a persistent burden in Nottingham over the past decade, with ocular surface diseases, contact lens wear and systemic immunosuppression being the most common risk factors. More than 50% of the patients with IK required hospitalisation for intensive treatment, highlighting the burden of the disease on the patients and the healthcare system. Poor clinical outcome was significantly affected by older age, large infiltrate size and poor presenting vision. The second part (Chapter 5 and Chapter 6) systematically examined the effectiveness and safety of adjuvant therapeutic corneal collagen cross-linking (PACK-CXL) and amniotic membrane transplant for treating IK, in addition to standard antimicrobial therapy. The meta-analyses demonstrated that both interventions significantly expedited the healing of IK, though the overall quality of evidence was low, highlighting the need for further high-quality randomised controlled trials. The third part (Chapter 7 and Chapter 8) highlighted a body of work in developing a new class of HDP-based antimicrobial therapy for IK based on hybrid derivatives of human cathelicidin (LL-37) and human beta-defensins-1 to -3. CaD23, derived from LL-37 and HBD-2, exhibited good in vitro efficacy against Gram-positive bacteria and moderate efficacy against Gram-negative bacteria. It demonstrated a rapid antimicrobial activity, which was likely attributed to its membrane-permeabilising activity, supported by SYTOX green dye uptake assay and molecular dynamics simulation study. CaD23 was also shown to exhibit a strong additive effect when used in combination with conventional antibiotics against Gram-positive bacteria. Finally, CaD23 exhibited good antimicrobial efficacy against Gram-positive bacteria (1.2 logCFU or 94% reduction in the bioburden) in a murine bacterial keratitis model. The discovery of CaD23 has provided a new scaffold for future development of newer generations of hybrid peptides

Influence of the gut microbiome on plasma metabolite patterns

The gut microbiome has become a burgeoning field of research and is proven to play a role in in many host physiological aspects, also contributing to the blood metabolome that is otherwise produced by tissues of the host organism. In order to increase the mechanistic understanding of how the microbiome influences its host health, there is a need to integrate knowledge of the composition of the gut microbiome with its functionality in terms of microbiota-mediated metabolic processes. The aim of this project was to obtain detailed insight in the mammalian-microbiome co-metabolism of endogenous metabolites and the extent to which the microbiome influences the plasma metabolome observed. This was achieved with the help of the MetaMap®Tox database in which the metabolome and toxicity data of more than 800 compounds are stored. To elucidate this interaction, metabolites produced by the gut microbiome in the blood of our animal model, Wistar rats, had to be determined. Therefore, antibiotics were used to modulate the microbial communities of Wistar-rats. After 28-day oral administration, metabolomics of plasma, feces, and cecum-content was done. Additionally, DNA was extracted from rat feces and the 16S subunit was sequenced to perform a core diversity analysis. Specific plasma metabolome patterns were established, and microbiome-related metabolites identified as key metabolites in MetaMap®Tox. Metabolites such as hippuric acid, indole derivates and glycerol appeared to be microbiome-derived or -associated plasma metabolites. In general, most changes were observed in metabolites belonging to the class of bile acids, complex lipids, fatty acids and related metabolites, as well as amino acids and related metabolites. Especially abrupt changes observed in the bile acid pool after antibiotic administration evidenced a strong influence of the microbiome on bile acid metabolism. In both feces and cecum-content, where almost all plasma metabolites could be determined as well, a treatment-related effect was observed, as well as only minimal, if any, differences between samples of male and female animals. The effects of the tested antibiotics, each possessing a different activity spectrum, could be separated from each other on the basis of the feces and cecum content metabolome. Largest changes were observed for the classes of lipids, bile acids and amino acids. It could also be shown that metabolome changes can be detected equally well in feces as in the cecum content, thus allowing to use a non invasive method for measurements of studies on metabolism by the gut microbiota. In conclusion, the functional microbial changes of the gut microbiome had to be assessed and the interactions between gut microbes and the host by applying metabolomics and taxonomic profiling had to be elucidated. The results of this thesis suggest that plasma and feces based metabolic profiling via a targeted analysis turned out to be a suitable tool to investigate the microbial functionality of the gut microbiome.</p