HIV infection begins when gp120 envelope protein on the viral surface binds to the CD4 receptor on the host cell. This initial protein-protein interaction starts the rest of the HIV lifecycle of coreceptor binding, fusion and replication. One of the targets of HIV entry inhibitors is the interaction between CD4 and gp120. A large percentage of CD4-gp120 contacts revolved around a cavity in gp120 in which the PHE43 residue of CD4 caps. We were able to design and synthesize a progression of small molecule compounds targeting the PHE43CD4 cavity in gp120 based on a previous CD4-mimetic, NBD-556. By either soaking or co-crystallizing the newly designed compounds bound to gp120, we were able to solve four x-ray crystal structures in order to observe the interactions with the binding cavity on the atomic level. Using x-ray crystal structure, isothermal calorimetry and viral binding assay to guide design, we were able to improve the binding affinity more than 30 fold compared to the original NBD-556. Our most potent compound DMJ-II-121-R,R is able to bind to gp120 at a Kd of 0.11 micromolar and specifically block HIV-1 entry at an IC50 of 2.3 micromolar. Along with improved potency, the new design alleviated the agonistic properties of the original NBD-556, which was inducing gp120 to bind to the coreceptors on the host cell instead of blocking the progression of the HIV lifecycle. In parallel, we also utilized the soakable gp120 crystal system to screen a library of 352 fragments of various shapes using x-ray crystallography to detect and identify two positive hits, benzimidazole and 3-hydroxyphenylacetic acid. The possible leads from the two identified fragments along with our improved potency of NBD-556 based derivatives offer valuable insight to guide us on the development toward a subnanomolar small molecular antagonist of gp120-CD4 binding.

The lymphatic vascular system is necessary for physiological regulation of tissue fluid homeostasis and absorption of dietary fat. Lymphatics also function in the inflammatory response and are involved in pathological conditions such as wound healing and cancer. We show that the Notch signaling pathway is a regulator of both developmental and pathological lymphangiogenesis. Notch1 and Notch4 are expressed by the lymphatic endothelium, and Delta-like ligand 4 (Dll4) is the predominantly expressed Notch ligand in the developing lymphatic vessels of the embryonic dermis and pathological lymphatic vessels of the wounded cornea. Dll4 was able to induce Notch activation in human dermal lymphatic endothelial cells (HDLECs), whereas Jagged1 (Jag1) was not. In HDLECs, Notch signaling is activated in response to Vascular Endothelial Growth Factor (VEGF) or Vascular Endothelial Growth Factor-C (VEGF-C) stimulation. In vitro assays demonstrated that Notch activation inhibits HDLEC proliferation, migration, and capillary network formation; these effects were coincident with increased levels of HEY1 and HEY2, biphasic regulation of VEGFR-3, and decreased levels of VEGFR-2. Using genetic intervention of Notch signaling, we demonstrated that Notch regulates developmental sprouting lymphangiogenesis by restricting growth and sprouting of lymphatics in the murine embryonic dermis. Using pharmacological intervention of Notch signaling, we found that Notch restricted pathological sprouting lymphangiogenesis in the corneal suture assay, which models inflammation-induced lymphangiogenesis. However, pharmacological intervention of Notch signaling did not measurably affect pathological sprouting lymphangiogenesis in an orthotopic tumor model of human breast cancer. Our data from analysis of HDLECs, dermis, and sutured cornea support a role for Dll4-driven Notch signaling in restricting sprouting lymphangiogenesis. Lymphatic specification/separation requires a venous endothelial cell to become a lymphatic endothelial cell, and lymphatic valve formation requires a duct endothelial cell to become a valve endothelial cell. Through analysis of genes regulated by Notch in HDLECs, we demonstrated that Notch determines lymphatic endothelial cell fates. Notch inhibits genes critical for lymphatic specification and separation (PROX1, PDPN), and induces genes important for lymphatic valve formation (FNEIIIA, ITGA9, CX37). We conclude that Notch is a context-dependent regulator of lymphangiogenesis. Notch functions in the tip/stalk, venous to lymphatic, and duct endothelial to valve endothelial cell fate decisions in lymphatic vasculature. Given the critical functions of the lymphatic vasculature in multiple physiological and pathological settings, understanding Notch functions in the lymphatic vasculature is critical to design treatments for conditions caused by lymphatic malfunction.

Many diseases are caused by pathogenic bacteria. A key example of this is sepsis, which is mostly caused by staphylococci and Gram-negative bacteria. In addition, the highly resistant ESKAPE pathogens are responsible for the majority of hospital acquired infections. In order to treat bacterial infections effectively, and to avoid promoting bacterial resistance against antibacterial drugs, the correct agents must be used, for which in turn the detection and identification of pathogenic strains is essential. This research aims to develop selective chromogenic culture media, by introducing new antibacterial agents for the improved selectivity and new chromogenic substrates for selective visualisation of certain bacterial strains. The intention of the major part of this work was to inhibit the growth of commensal bacteria in clinical samples, as they mask the growth of the infection-causing bacteria. New and known compounds were prepared for 3 evaluation as alanine racemase inhibitors. The compounds were tested on a range of clinical pathogenic and non-pathogenic bacterial strains. The molecules developed were based on the amino acid alanine and utilised bioisosteres and other replacements for the carboxylic acid moiety. Key compounds targeted included alanylmethanesulfonamide 27-L, 1-aminoethyl5-oxo-1,2,4-oxadiazole 33-L and 1-aminoethyltetrazole 32a-L. Each compound was tested initially as the alanyl-X dipeptide form. While most of the alanine bioisosteres were known structures, their novel peptide derivatives required synthetic development using both solution and solid phase techniques. The solid phase synthesis of several C-terminal 1aminoethyltetrazole peptides was successfully established by using 2-chlorotrityl chloride resin. The investigation of the antimicrobial activity of the synthesised compounds identified several clinically applicable selective inhibitors. These compounds were shown to provide differentiation between Salmonella and Escherichia coli, or enterococci and streptococci. This work also gave a useful comparison between the different alanine bioisosteres, and showed the importance of di- and oligopeptide permease systems in order to reach sufficient bacterial activity. The microbiological activity of 1- aminoethyltetrazole peptide derivatives was studied in more detail, due to their potential in clinical applications for the diagnosis of food poisoning. In other work, also directed towards the rapid and selective detection and identification of pathogenic bacteria in a clinical environment, new chromogenic substrates were prepared. Each of these compounds contained a chromogen with a phenoxazin-3-one scaffold linked to an amino acid residue. The purpose of the amino acid is to act as a unit recognised and cleaved by specific hydrolytic bacterial enzymes. Upon liberation, electronic differences between the conjugated and free forms of the chromogen resulted in the development of distinct colour changes, which provide the basis of 4 bacterial detection and identification. Synthetic methods have been developed for the efficient and economical production of this series of substrates. After preparation, these compounds were tested against a panel of clinically relevant bacteria. The aim of these substrates was to present an alternative substrate for (N-β-alanyl)-7-amino-1-pentylphenoxazine-3-one 86a, which is applied commercially in chromID® Pseudomonas aeruginosa chromogenic medium designed for the clinical detection of P.aeruginosa. The new substrates are designed to fully explore the chemical space of phenoxazinonebased chromogenic substrates, and to decrease the colour, as substrate 86a causes significant background colour in culture media. The future application of these substrates in chromogenic media resides in their potential to advance the identification of specific pathogenic bacteria and to thus facilitate the treatment of bacterial infections.

Amorphous solid dispersion (ASD) of a poorly soluble active pharmaceutical ingredient (API) in a polymeric matrix is a promising approach to increase the solubility, dissolution rate and hence bioavailability of the API. From an industrial point of view, spray-drying represents the main solvent evaporation process used for the manufacture of solid dispersions. The aim of the present PhD thesis was to evaluate the accuracy of: i) thermodynamic models (e.g. solubility parameter and Flory-Huggins theories), ii) standard screening methodologies (e.g. solvent casting and quench cooling) and iii) novel screening approaches for predicting the miscibility of binary spray-dried solid dispersions (SDSDs) so that the best performing API-polymer systems at adequate drug-loading (DL) can be selected. Two novel approaches for screening improvement, miniaturization and downscaling of regular spray-drying, were investigated and applied to API-polymer systems consisting of models drugs (Ibuprofen, Naproxen, Carbamazepine and Itraconazole) and seven polymers at four DLs. Screened samples were characterized using modulated differential scanning calorimetry (mDSC), X-ray powder diffraction (XRPD), thermo-gravimetric analysis (TGA) and scanning electron microscopy (SEM). Results obtained from miscibility and solid state characterization were compiled into principal components analysis (PCA) in order to qualitatively rank the screening approaches based upon their prediction accuracy. Non-sink dissolutions conditions with regard to the crystalline API were performed to assess the solubility enhancement and the extent of supersaturation of screened samples. The two proposed screening approaches were found to provide a greater accuracy than traditional screening methodologies to predict the miscibility of SDSDs. The limitations of theoretical models and standard screening methods tested are symptomatic of the gap existing between equilibrium solid solubility to kinetic miscibility as well as the importance of the preparation method with regard to ASD properties. Therefore, the main benefits of the novel approaches rely on their capacity to better reproduce the operating mode and process conditions of regular spray-dryer, while minimizing API needs for a production, significantly. The outcome of this work favours the downscaling approach due to its improved ability to ease the transfer from screening phases to manufacturing stage in the selection of adequate polymer and DL. In this regard, a novel three-stage decision protocol that implements spray-drying in a methodical small-scale approach for the development of ASDs during preclinical activities was developed and has successfully replaced all former practices in UCB projects.

Background: Influenza-like illness (ILI) and asthma are common diagnoses in the general practice and exert considerable morbidity and mortality worldwide. One potentially important strategy to reduce this is to determine whether commonly prescribed drugs modify the occurrence and outcomes of respiratory diseases including mortality. The benzodiazepines, a class of psychoactive drugs generally used in the treatment of anxiety and sleep disorders, have recently been suggested as having detrimental effects on immune response to infection, predisposing its users to increased risk of infection and mortality. These drugs have also been implicated in the pathogenesis of asthma. This thesis therefore aimed to investigate whether benzodiazepines modify the occurrence of asthma exacerbation and subsequent mortality in patients with asthma as well as ILI-related pneumonia and ILI-related mortality in patients with ILI. Methods: The Clinical Practice Research Datalink (CPRD) was used as the data source. CPRD contains the medical records of over 13 million patients prospectively collected from over 600 general practices across the United Kingdom and has linkages to the Hospital Episode Statistics database [HES] and national death registry data (the Office of National Statistics [ONS]) which were utilised in this study. CPRD-HES linked data was used to validate diagnoses of asthma exacerbation whereas ONS mortality data validated deaths identified from CPRD. Case-control and cohort study designs were used to investigate associations between benzodiazepines and the occurrence of asthma exacerbation, ILI-related pneumonia and mortality. Results: After adjusting for a wide range of potential confounders including physical and psychiatric comorbidities, and concurrent use of other drugs, exposure to benzodiazepines was associated with statistically significant increased occurrence of asthma exacerbation, ILI-related pneumonia and mortality. These associations were observed with both short term and chronic benzodiazepines use. However, the effect of individual benzodiazepines varied across the outcomes of interest with some of the associations lacking statistical significance. For instance, current use of diazepam and temazepam but not lorazepam showed statistically significant associations with increased occurrence of asthma exacerbation. Conclusion: Overall, findings of this research signal an adverse benzodiazepine effect and hence suggest that a precautionary approach should be exercised when prescribing benzodiazepines in the interim before conclusive evidence is yielded by further research, especially in patients who may already be at increased risk of asthma exacerbation or pneumonia and mortality.

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