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The amino acid sequence of a protein encodes its folding, the reaction by which a peptide self-assembles into its native functional shape. A folded protein will then go on to carry on biological functions such as ligand binding, signaling, mechanical functions, or biomolecular catalysis. While much experimental work has been done to elucidate protein structure and conformational dynamics, Molecular Dynamics (MD) simulations have been necessary to provide atomic-level details of biomolecular dynamics. A challenge in using MD is the the large computational cost required to reach biologically relevant timescales when integration steps are typically limited to a few femtoseconds. To address this challenge, specialized hardware such as the ANTON supercomputer or distributed computing platforms like Folding@home can be used to collect milliseconds of aggregate trajectory data. From these datasets, kinetic network models called Markov state models (MSMs) can be constructed to infer long timescale dynamics from ensembles of short trajectories. These models can be analyzed in a human interpretable way and make physics-based connections to experimental observables. This dissertation describes how we have used MD simulations and MSMs to model protein folding reactions and protein-protein binding reactions to better understand mutation effects on our systems of interest. The first chapter of this dissertation describes MD simulations FOXO1 FKH domain folding, which we used MSMs to characterize at atomic resolution. To predict how mutations found in diffuse large B-cell lymphoma (DLBCL) cell lines effect protein stability, we developed an MSM-based hydrophobic free energy of transfer (HT) model to estimate mutation effects. Our HT model results agree better with experiment than other state-of-the-art computational methods. Chapter two describes how we have used approximately 43000 relative binding free energy calculations via the expanded ensemble (EE) method to perform in silico site saturation mutagenesis on miniprotein binders to the highly conserved influenza A H1 hemagglutinin stem region (HA2) de novo designed by the Baker [66]. These miniproteins were selected through an exhaustive design process with iterations of computational design, high throughput affinity screens, and site saturation mutagenesis. We compare our EE SSM method with inferred relative affinities from Chevalier et al.[66], as well as with the state-of-the-art Rosetta method Flex ddG. While Flex ddG predictions are more accurate on average, they are highly conservative. In contrast, EE predictions can better classify stabilizing and destabilizing mutations. We also use a Shannon entropy based method to identify residue positions that are more susceptible to mutation. This work suggests that simulation-based free energy methods can provide predictive information for in silico affinity maturation of designed miniproteins, with many feasible improvements to the efficiency and accuracy within reach. In the final chapter, we atttempt to model the complete binding reactions of the 6 miniproteins mentioned above. We used unbiased simulations to build standard msms and , in combination with biased simulations, multiensemble markov models (MEMMs) of binding for each wild type and affinity matured pair. The unbiased MSMs show that the affinity matured miniproteins prefer different bound states than the wild type miniproteins. Additionally, they provide physically realistic k_on_s and a macroscopic 3 state pathway through an encounter complex. We characterize each of those states and use an contact map based structural similarity index measure (SSIM) and residue-wise Kullback-Leibler divergence method to better understand the differences in the bound states between affinity matured and wild type construct. Interestingly, while our biased simulations do see unbinding transitions, in estimating the MEMM, they overweight the unbinding reaction and unbound state, leading to models that do not make physical sense. This demonstrates that more sensitive enhances sampling techniques may be necessary for building MEMMs. The final two chapters of this dissertation present new methodologies for computational protein design, making great strides towards a dynamic understanding of how proteins bind their targets and how mutations effect those reactions. / Chemistry

Viral proteases play crucial roles in the life cycle and maturation of many viruses by processing the viral polyprotein after translation and in some cases cleaving host proteins associated with the immune response. The essential role of viral proteases makes them attractive therapeutic targets. In this thesis, I provide an introductory summary of viral proteases, their structure, mechanism, and inhibition, while the breadth of this thesis focuses on the Hepatitis C virus (HCV) NS3/4A and Zika virus (ZIKV) NS2B/NS3 viral proteases. HCV NS3/4A protease inhibitors (PIs) have become a mainstay in combination therapies. However, drug resistance remains a major problem against these PIs. In this thesis, I applied insights from the HCV substrate envelope (SE) model to develop strategies for designing PIs that are less susceptible to resistance. Also, I used the HCV NS3/4A protease as a model system to decipher the molecular mechanism and role of fluorination in HCV PIs potency and drug resistance. The drug design strategies described in this thesis have broad applications in drug design. The ZIKV is an emerging global threat, and currently, with no treatment available. In this thesis, I described the discovery, biochemical and antiviral evaluation of novel noncompetitive quinoxaline-based inhibitors of the ZIKV NS2B/NS3 protease. The inhibitors are proposed to interfere with NS2 binding to NS3, thereby preventing the protease from adopting the closed and active conformation. The inhibitors from this work will serve as lead compounds for further inhibitor development toward the goal of developing antivirals.

Lung cancer is a leading cause of cancer-related mortality irrespective of gender. The Sulfiredoxin (Srx) and Peroxiredoxin (Prx) are a group of thiol-based antioxidant proteins that plays an essential role in non-small cell lung cancer. Understanding the molecular characteristics of the Srx-Prx interaction may help design the strategies for future development of therapeutic tools. Based on existing literature and preliminary data from our lab, we hypothesized that the Srx plays a critical role in lung carcinogenesis and targeting the Srx-Prx axis or Srx alone may facilitate future development of targeted therapeutics for prevention and treatment of lung cancer. First, we demonstrated the oncogenic role of Srx in urethane-induced lung carcinogenesis in genetically modified FVB mice. The Srx-null mice showed resistance to urethane-induced lung cancer. Second, we demonstrated the Srx and Prx sites important for Srx-Prx interaction. The orientation of this arm is demonstrated to cause some steric hindrance for the Srx-Prx interaction as it substantially reduces the rate of association between Srx and Prx. Finally, we carried out virtual screening to identify molecules that can successfully target Srx-Prx interaction. Multiple in-silico filters were used to minimize the number of chemicals to be tested. We identified ISO1 as an inhibitor of the Srx-Prx interaction. KD value for Srx-ISO1 interaction is calculated to be 42 nM. Together, these data helps to identify an inhibitor (ISO1) of the Srx-Prx interaction that can be further pursued to be developed as a chemotherapeutic tool.

Metal-based engineered nanomaterials (ENMs) have potential to revolutionize diagnosis, drug delivery and manufactured products, leading to greater human ENM exposure. It is crucial to understand ENM pharmacokinetics and their association with biological barriers such as the blood-brain barrier (BBB). Physicochemical parameters such as size and surface modification of ENMs play an important role in ENM fate, including their brain association. Multifunctional ENMs showed advantages across the highly regulated BBB. There are limited reports on ENM distribution among the blood in the brain vasculature, the BBB, and brain parenchyma. In this study, ceria ENM was used to study the effect of size on its pharmacokinetics. Four sizes of ceria ENMs were studied. Five nm ceria showed a longer half-life in the blood and higher brain association compared with other sizes and 15 and 30 nm ceria had a higher blood cell association than 5 or 55 nm ceria. Because of the long circulation and high brain association of 5 nm ceria compared with other sizes, its distribution between the BBB and brain parenchyma was studied. The in situ brain perfusion technique showed 5 nm ceria (99%) on the luminal surface of the BBB rather than the brain parenchyma. For biomedical applications in the central nervous system (CNS), it is vital to develop stable and biocompatible ENMs and enhance their uptake by taking advantage of their unique properties. Cross-linked nanoassemblies entrapping iron oxide nanoparticles (CNA-IONPs) showed controlled particle size in biological conditions and less toxicity in comparison to Citrate-IONPs. CNA-IONPs considerably enhanced MRI T2 relaxivities and generated heat at mild hyperthermic temperatures (40 ~ 42°C) in the presence of alternating magnetic field (AMF). Numerous researchers showed mild whole body hyperthermia can increase BBB permeability for potential brain therapeutic application. Compared to conventional hyperthermia, AMF-induced hyperthermia increased BBB permeability with a shorter duration of hyperthermia and lower temperature, providing the potential to enhance IONP flux across the BBB with reduced toxicity. Overall, ENMs with optimized physicochemical properties can enhance their flux across the BBB into the brain with desirable pharmacokinetics, which provide great potential for diagnosis and therapy in the CNS.

The overall objectives of this research project were i) to develop and evaluate methods of characterizing nasal spray products using realistic nasal airway models as more clinically relevant in vitro tools and ii) to develop and evaluate a novel high-efficiency antibiotic nanoparticle dry powder formulation and delivery device. Two physically realistic nasal airway models were used to assess the effects of patient-use experimental conditions, nasal airway geometry and formulation / device properties on the delivery efficiency of nasal spray products. There was a large variability in drug delivery to the middle passages ranging from 17 – 57 % and 47 – 77 % with respect to patient use conditions for the two nasal airway geometries. The patient use variables of nasal spray position, head angle and nasal inhalation timing with respect to spray actuation were found to be significant in determining nasal valve penetration and middle passage deposition of Nasonex®. The developed test methods were able to reproducibly generate similar nasal deposition profiles for nasal spray products with similar plume and droplet characteristics. Differences in spray plume geometry (smaller plume diameter resulted in higher middle passage drug delivery) were observed to have more influence on regional nasal drug deposition than changes to droplet size for mometasone furoate formulations in the realistic airway models. Ciprofloxacin nanoparticles with a mean (SD) volume diameter of 120 (10) nm suitable for penetration through mucus and biofilm layers were prepared using sonocrystallization technique. These ciprofloxacin nanoparticles were then spray dried in a PVP K30 matrix to form nanocomposite particles with a mean (SD) volume diameter of 5.6 (0.1) µm. High efficiency targeted delivery of the nanocomposite nasal powder formulation was achieved using a modified low flow VCU DPI in combination with a novel breathing maneuver; delivering 73 % of the delivered dose to the middle passages. A modified version of the nasal airway model accommodating Transwell® inserts and a Calu-3 monolayer was developed to allow realistic deposition and evaluation of the nasal powder. The nanocomposite formulation was observed to demonstrate improved dissolution and transepithelial transport (flux = 725 ng/h/cm2) compared to unprocessed ciprofloxacin powder (flux = 321 ng/h/cm2).

α7 Neuronal nicotinic acetylcholine receptors are one of two major classes of receptors responsible for cholinergic neurotransmission in the central nervous system. The existence of α7 neuronal nAChRs in different regions of the nervous system suggests their involvement in certain essential physiological functions as well as in disorders such as Alzheimer’s disease (AD), drug dependence, and depression. This project was aimed toward the discovery and development of small–molecule arylguanidines that modulate α7 nAChR function with improved subtype-selectivity through an allosteric approach. Identifying the required structural features of these small molecules allowed optimization of their negative allosteric modulator (NAM) actions at α7 neuronal nAChRs. MD-354 (3-chlorophenylguanidine) was the first small–molecule NAM at α7 nAChRs; however, it also binds at 5-HT3 receptors. The N-methyl analog of MD-354 appeared to be more selective toward α7 nAChRs than 5-HT3 receptors. Comparative studies using two series of novel compounds based on MD-354 and its N-methyl analog explored the aryl 3-position and investigated whether or not the MD-354 series and the N-methyl series bind in the same manner. Biological potencies of the MD-354 series and the N-methyl series of compounds, obtained from electrophysiological assays with Xenopus laevis oocytes expressing human α7 nAChRs in two-electrode voltage-clamp assays, showed that N-(3-iodophenyl)-N- methylguanidine (28) is the most potent analog at α7 nAChRs. Our comparative study and Hansch analyses indicated different binding modes of the two series. In addition, we investigated: i) the length/size of the aliphatic side chain at the anilinic nitrogen, ii) the effect of alkylating the guanidine nitrogen atoms, and iii) the necessity of the presence of these nitrogen atoms for the inhibitory effects of arylguanidines at α7 nAChRs. In efforts to explain the varied functional activity of these arylguanidines, homology models of the extracellular domain and the transmembrane domain of human α7 nAChRs were developed, allosteric sites identified, and docking studies and hydropathic analysis conducted. The 3D quantitative structure-activity relationships for our compounds were also analyzed using CoMFA. A pharmacophore for arylguanidines as α7 nAChR NAMs was identified. Together, these data should be useful for the subsequent design of novel arylguanidine analogs for their potential treatment of neurological disorders.

Dans la recherche de nouveaux traitements des maladies broncho-pulmonaires comme l'asthme et la broncho-pneumopathie chronique obstructive, les inhibiteurs de PDE4 sont des cibles intéressantes. Dans cette voie, notre laboratoire s'intéresse à la synthèse d'une nouvelle famille d'inhibiteurs à base pyridazinone. Pour cela, cette thèse couple la modélisation moléculaire (docking : développement méthodologique et application), la synthèse organique ainsi que des tests biologiques.Dans un premier temps, le développement du logiciel d'amarrage moléculaire AlgoGen a été poursuivi. AlgoGen (créé initialement à l'université de Lorraine en 2009) est un logiciel qui couple un algorithme génétique pour la recherche conformationnelle à une évaluation de l'énergie protéine-ligand à un niveau quantique semi-empirique alors que les autres logiciels existant effectuent ce calcul à un niveau classique en général. Le calcul d'une énergie à un niveau quantique est très coûteux en temps. C'est pourquoi, nous avons apporté de nombreuses modifications à ce logiciel afin d'accroître son efficacité dans la recherche conformationnelle. Ce logiciel a ensuite été utilisé sur un jeu de 22 dimères (typiques des reconnaissances moléculaires biologiques) et à huit systèmes protéine-ligand.Dans un deuxième temps, huit inhibiteurs ont été synthétisés et testés in vitro sur la cible PDE4. Pour compléter ce volet expérimental, une étude de structure-activité a été effectuée grâce au docking moléculaire (AlgoGen, Autodock, Glide) pour rationaliser les activités mesurées (IC50). Pour terminer, des pharmaco-modulations guidées par docking ont été réalisées afin de proposer de nouveaux inhibiteurs de plus grandes affinités avec la protéine PDE4D. / For the research of new treatment of bronchopulmonary diseases such as asthma and chronic obstructive pulmonary disease (COPD), the PDE4 inhibitors are an attractive target. Our laboratory is interested in a new PDE4 inhibitors family based on the pyridazinone pattern. For this purpose, this thesis couples molecular modeling (docking: methodological development and application), organic synthesis and biological tests.First, the development of the molecular docking software AlgoGen was continued. AlgoGen (initially created at university of Lorraine in 2009) is a program which couples a genetic algorithm for the conformational research and a protein-ligand energy evaluation at the quantum semi-empirical level while other software do this evaluation at a classical level. Quantum energy calculations are very time consuming. That is the reason why some modifications have been made to improve its efficiency for the conformational search. This software was then used for calculations on a set of 22 dimers (typical in biological molecular recognition) as well on 8 ligand-protein complexes.Secondly, eight inhibitors were synthesized and tested in vitro on the PDE4 target. To complete the experimental part, a structure-activity relationship study was carried out through a molecular docking to rationalize the measured activity (IC50). Finally, pharmaco-modulations guided by docking were made to propose new inhibitors with more affinity with the protein.

La dépression est l’un des troubles mentaux les plus fréquents de nos jours. C’est une maladie liée en général à un déficit en neurotransmetteurs monoaminergiques (sérotonine, noradrénaline et dopamine). Les antidépresseurs actuels agissant via des mécanismes monoaminergiques présentent de nombreux effets secondaires et peuvent conduire à une accoutumance. L’une des approches impliquerait le ciblage des récepteurs mélatoninergiques afin de resynchroniser les rythmes circadiens qui sont perturbés dans certaines pathologies du système nerveux central, notamment la dépression. La conception de ligands non monoaminergiques et possédant des propriétés chronobiotiques constituerait une stratégie prometteuse.L’agomélatine (Valdoxan®) issue d’une collaboration entre le laboratoire de chimie thérapeutique (EA4481 - GRIIOT) et les laboratoires Servier est commercialisée depuis 2009 pour le traitement de la dépression majeure. Ce bioisoster naphtalénique de la mélatonine possède un mécanisme d’action innovant. Elle se distingue par ses propriétés agoniste non sélectif des récepteurs mélatoninergiques MT1 et MT2 et antagoniste des récepteurs 5-HT2c.L’objectif de ce travail réside dans la conception et la synthèse de nouveaux ligands successeurs de l’agomélatine présentant un profil pharmacologique et pharmacocinétique amélioré. Différentes pharmacomodulations ont été réalisées sur l’agomélatine. Les stratégies de «Drug Design» notamment le principe de bioisostérie ont été appliquées, nous permettant ainsi la synthèse de nouvelles familles de composés présentant des profils pharmacologiques intéressants. / Nowadays, depression related to a deficit in nonoaminergic neurotransmitters, is the most frequent mental illness. Available antidepressive drugs acting through monoamnergic mechanisms possess a lot of side effects and can lead to an addiction. One approach involves targeting melatoninergic receptors to resynchronize circadian rhythms, which are known to be perturbed in some pathology related to nervous central system as depression. So conception of non-monoaminergic ligands with chronobiotic properties would constitute a promising strategy.Agomelatine (Valdoxan®) a novel antidepressant developed by Servier and our laboratory (EA4481 - GRIIOT) was granted marketing authorization in 2009 for the treatment of major depressive disorder. This naphthalen analogous of melatonin possess an innovative mechanism. It acts as a non selective melatoninergic MT1/MT2 receptors agonist and a serotonin 5-HT2c receptor antagonist.The aim of this work is to design and synthesize new potential successors of agomelatine with an improved pharmacological and pharmacokinetic profile. Drug Design strategies such as bioisosterism were applied to allow the elaboration of new series of compounds with interesting pharmacological profiles.