Στα πλαίσια της διπλωματικής εργασίας μελετήθηκαν οι αλγόριθμοι και τα εργαλεία σχεδίασης Φαρμάκων με τη βοήθεια Η/Υ, που υπάρχουν στη διεθνή βιβλιογραφία. Η μελέτη αναφέρθηκε σε αλγορίθμους «εξ αρχής» σχεδιασμού φαρμακοφόρων μορίων (de novo drug design). Στόχος της διπλωματικής εργασίας ήταν η σχεδίαση ενός πλαισίου αξιολόγησης των σχετικών εργαλείων και αλγορίθμων σχεδιασμού Φαρμάκων με τη βοήθεια Η/Υ και η αναφορά πιθανών βελτιώσεων στα ανοικτά προβλήματα που προκύπτουν. / The thesis was about algorithms and tools for drug design with the use of computers, algorithms and tools which already exist in the international bibliography. The study referred to algorithms for de novo drug design. The aim of the thesis was the design of a frame for the evaluation of the relevant tools and algorithms and also the report of possible improvements to the open issues that arise.

The central dogma of molecular biology outlines the process of information transfer from a DNA sequence, to a protein chain. Beyond the step of protein synthesis, there are a variety of post-translational modifications that can take place, one of which is addition of carbohydrate chains to nascent proteins, known as glycosylation. The N-linked glycosylation pathway is responsible for the covalent attachment of multifunctional carbohydrate chains on asparagine residues of nascent proteins at Asn-X-Ser/Thr consensus sequences. These carbohydrate chains are thought to aid in cell signaling, immune recognition, and other processes. Golgi alpha-mannosidase II (GMII) is the enzyme in the N-glycosylation pathway that is responsible for cleaving two mannose linkages in the oligosaccharide GnMan5Gn2 (where Gn is N-acetylglucosamine and Man is mannose), thereby producing GnMan3Gn2 , which is the committed step in complex N-glycan synthesis. It has been speculated that GMII is an excellent therapeutic target for cancer treatment, as the unusual distribution of carbohydrates on the surface of tumour cells has been characterized in many cancers. In addition, swainsonine-—a strong, yet nonspecific inhibitor of GMII—-has been shown to block metastasis and improve the clinical outcome of patients with certain cancers, including those of the colon, breast and skin. This thesis examines Golgi alpha-mannosidase II from Drosophila melanogaster (dGMII) as a model for all GMII enzymes. First, a 1.80 Angstrom resolution crystal structure of a weak inhibitor, kifunensine, binding to dGMII provides mechanistic insights into the substrate distortion in the GMII reaction. It is hypothesized that the GMII reaction proceeds via a 1 Sinterintermedi-ate. Second, a 1.40 Angstrom resolution structure of a mutant dGMII bound to its natural substrate, GnMan5Gn, identifies key substrate binding and catalytic residues, as well as expanding the definition of the GMII active site to include two distant sugar−binding subsites. Finally, the results are taken together, with knowledge of other related enzymes to synthesize a plausible itinerary for the GMII reaction.

Computer-aided drug design is an exceptionally useful tool for screening a large number of potential drug molecules to evaluate their therapeutic potential. This technique is both effective and economical. Approximately 120 imidazole-containing molecules were computationally designed and evaluated using gas-phase and solution-phase simulations to assess their propensity for acting as a chelating agent with twenty-six biologically relevant cations. Of particular interest was their ability to chelate Zn2+ and Ca2+, which play a key role in the degradation of neurons following an ischemic stroke. The ultimate goal was to design a small molecule that could be administered before a medical procedure that featured stroke as a possible side effect. In the event that a stroke occurred, the destruction of neurons caused by release of excess Ca2+ and Zn2+ would be diminished and the patient would maintain motor and cognitive function. Promising in silico results were obtained.

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Cocaine is a widely abused drug without an FDA-approved medication. It has been recognized as an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. butyrylcholinesterase (BChE)-catalyzed hydrolysis. However, the native BChE has a low catalytic activity against cocaine. We recently designed and discovered a set of BChE mutants with a high catalytic activity specifically for cocaine. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against cocaine, but also certain selectivity for cocaine over neurotransmitter acetylcholine (ACh) such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. Through integrated computational-experimental studies, several BChE mutants were identified to have not only a considerably improved catalytic efficiency against cocaine, but also the desirable selectivity for cocaine over ACh. Representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg, LD100). Pretreatment with the BChE mutant (i.e. 1 min prior to cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. The in vivo data reveal the primary factor, i.e. the relative catalytic efficiency, determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment. For further characterization in animal models, we successfully developed high-efficiency stable cell lines efficiently expressing the BChE mutants by using a lentivirus-based repeated-transduction method. The large-scale protein production enabled us to further characterize the in vivo profiles of the BChE mutant concerning the biological half-life and potency in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant can rapidly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.

Cocaine is one of the most reinforcing drugs of abuse and has caused serious medical and social problems. There is no FDA-approved medication specific for cocaine. It is of a high priority to develop an effective therapeutic treatment for cocaine abuse. Human butyrylcholinesterase (BChE) has been recognized as a promising candidate of enzyme therapy to metabolize cocaine into biologically inactive metabolites and prevent it from reaching central nervous system (CNS). However, the catalytic activity of wide-type human BChE against cocaine is not sufficiently high for treatment of cocaine abuse. Dr. Zhan’s lab has successfully designed and discovered a series of high-activity mutants of human BChE specific for cocaine metabolism. This dissertation is mainly focused to address the possible concerns in further development of promising human BChE mutants for cocaine detoxification, including whether the administration of this exogenous enzyme will affect the cholinergic system, whether it can efficiently hydrolyze cocaine’s toxic metabolites, and whether the commonly used therapeutic agents will significantly affect the catalytic activity of the BChE mutants against cocaine when they are co-administered. According to the results obtained, all of the examined BChE mutants have a considerably improved catalytic efficiency against (-)-cocaine, without significantly improving the catalytic efficiency against any of the other examined substrates, including neurotransmitter acetylcholine. Two representative mutants (including E12-7) also have a considerably improved catalytic activity against cocaethylene (formed from combined use of cocaine and alcohol) compared to wild-type BChE, and E12-7 can rapidly metabolize cocaethylene, in addition to cocaine, in rats. Further evaluation of possible drug-drug interactions between E12-7 and some other commonly used therapeutic agents revealed that all of the examined agents, except some tricyclic antidepressants, do not significantly inhibit E12-7. In addition, an effort to discover new mutants with further improved activity against cocaine led to the discovery of a new BChE mutant, denoted as E20-7, according to both the in vitro and in vivo assays. The encouraging outcomes of the present investigation suggest that it is possible to develop a more effective enzyme therapy for cocaine abuse treatment using one of the most promising BChE mutants, such as E12-7 or E20-7.

Supersaturating Drug Delivery Systems (SDDS) could enhance oral bioavailability of poorly water soluble drugs (PWSD). Precipitation inhibitors (PIs) in SDDS could maintain supersaturation by inhibiting nucleation, crystal growth, or both. The mechanisms by which these effects are realized are generally unknown. The goal of this dissertation was to explore the mechanisms underpinning the effects of model PIs including hydroxypropyl β-cyclodextrins (HP-β-CD), hydroxypropyl methylcellulose (HPMC), and polyvinylpyrrolidone (PVP) on the crystal growth of indomethacin, a model PWSD. At high degrees of supersaturation (S), the crystal growth kinetics of indomethacin was bulk diffusion-controlled, which was attributed to a high energy form deposited on the seed crystals. At lower S, indomethacin growth kinetics was surface integration-controlled. The effect of HP-β-CD at high S was successfully modeled using the reactive diffusion layer theory. The superior effects of PVP and HPMC as compared to HP-β-CD at high S were attributed to a change in the rate limiting step from bulk diffusion to surface integration largely due to prevention of the high energy form formation. The effects of PIs at low S were attributed to significant retardation of the surface integration rate, a phenomenon that may reflect the adsorption of PIs onto the growing surface. PVP was selected to further understand the relationship between adsorption and crystal growth inhibition. The Langmuir adsorption isotherm model fit the adsorption isotherms of PVP and N-vinylpyrrolidone well. The affinity and extent of adsorption of PVP were significantly higher than those of N-vinylpyrrolidone, which was attributed to cooperative interactions between PVP and indomethacin. The extent of PVP adsorption on a weight-basis was greater for higher molecular weight PVP but less on a molar-basis indicating an increased percentage of loops and tails for higher molecular weight PVPs. PVP significantly inhibited indomethacin crystal growth at high S as compared to N-vinylpyrrolidone, which was attributed to a change in the growth mechanism resulting in a change in the rate limiting step from bulk diffusion to surface integration. Higher molecular weight PVPs were better inhibitors than lower molecular weight PVPs, which was attributed to a greater crystal growth barrier provided by a thicker adsorption layer.

Advances in anti-cancer drug development have vastly improved cancer treatment strategies over the past few decades. Chemotherapeutic agents are now being replaced with targeted therapies that have much greater potency and far fewer unpleasant side effects. At the center of this, cell signaling pathways have been targeted as they moderate gene expression, control proliferation and are often dysregulated in cancer. The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes in response to extracellular signals. Of the seven mammalian members described to date, Stat3 has received particular attention, as it regulates the expression of genes involved in a variety of malignant processes including proliferation, survival, migration and drug resistance. Aberrant Stat3 activation has been observed in a number of human cancers, and its inhibition has shown promising anti-tumour activity in cancer cells with elevated Stat3 activity. Thus, Stat3 has emerged as a promising target for the development of cancer therapeutics. While Stat3 signaling can be inhibited by targeting upstream regulators of Stat3 activation (such as Janus kinase 2), direct inhibition of Stat3 protein may offer improved response, larger therapeutic windows for treatment and fewer side effects. The work presented within this thesis is focused on optimizing known Stat3 inhibitor S3I-201, a small molecule Stat3 SH2 domain binder that was discovered in 2007. We have performed an extensive structure activity relationship study that has produced some of the most potent Stat3 inhibitors in the scientific literature. These compounds showed high-affinity binding to Stat3’s SH2 domain, inhibited intracellular Stat3 phosphorylation and selectively induced apoptosis in a number of cancer cell lines. Lead agents further inhibited tumour growth in xenograft models of human malignancies and had favourable pharmacokinetic and toxicity profiles.

Advances in anti-cancer drug development have vastly improved cancer treatment strategies over the past few decades. Chemotherapeutic agents are now being replaced with targeted therapies that have much greater potency and far fewer unpleasant side effects. At the center of this, cell signaling pathways have been targeted as they moderate gene expression, control proliferation and are often dysregulated in cancer. The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes in response to extracellular signals. Of the seven mammalian members described to date, Stat3 has received particular attention, as it regulates the expression of genes involved in a variety of malignant processes including proliferation, survival, migration and drug resistance. Aberrant Stat3 activation has been observed in a number of human cancers, and its inhibition has shown promising anti-tumour activity in cancer cells with elevated Stat3 activity. Thus, Stat3 has emerged as a promising target for the development of cancer therapeutics. While Stat3 signaling can be inhibited by targeting upstream regulators of Stat3 activation (such as Janus kinase 2), direct inhibition of Stat3 protein may offer improved response, larger therapeutic windows for treatment and fewer side effects. The work presented within this thesis is focused on optimizing known Stat3 inhibitor S3I-201, a small molecule Stat3 SH2 domain binder that was discovered in 2007. We have performed an extensive structure activity relationship study that has produced some of the most potent Stat3 inhibitors in the scientific literature. These compounds showed high-affinity binding to Stat3’s SH2 domain, inhibited intracellular Stat3 phosphorylation and selectively induced apoptosis in a number of cancer cell lines. Lead agents further inhibited tumour growth in xenograft models of human malignancies and had favourable pharmacokinetic and toxicity profiles.

Molecular modeling has come a long way during the past decades and in the current thesis modeling of biological membranes is the focus. The main method of choice has been classical Molecular Dynamics simulations and for this technique a model Hamiltonian, or force field (FF), has been developed for lipids to be used for biological membranes. Further, ways of more accurately simulate the interactions between solutes and membranes have been investigated. A FF coined Slipids was developed and validated against a range of experimental data (Papers I-III). Several structural properties such as area per lipid, scattering form factors and NMR order parameters obtained from the simulations are in good agreement with available experimental data. Further, the compatibility of Slipids with amino acid FFs was proven. This, together with the wide range of lipids that can be studied, makes Slipids an ideal candidate for large-scale studies of biologically relevant systems. A solute's electron distribution is changed as it is transferred from water to a bilayer, a phenomena that cannot be fully captured with fixed-charge FFs.  In Paper IV we propose a scheme of implicitly including these effects with fixed-charge FFs in order to more realistically model water-membrane partitioning. The results are in good agreement with experiments in terms of free energies and further the differences between using this scheme and the more traditional approach were highlighted. The free energy landscape (FEL) of solutes embedded in a model membrane is explored in Paper V. This was done using biased sampling methods with a reaction coordinate that included intramolecular degrees of freedom (DoF). These DoFs were identified in different bulk liquids and then used in studies with bilayers. The FELs describe the conformational changes necessary for the system to follow the lowest free energy path. Besides this, the pitfalls of using a one-dimensional reaction coordinate are highlighted.