La distribution de principes actifs dans le système nerveux central (SNC) est entravée par la présence d'une barrière physiologique, la barrière hémato-encéphalique (BHE). L'endothélium cérébral est pourvu d'un large éventail de systèmes de transport, parmi lesquels la trancytose récepteur-dépendante, qui peut être mise à profit pour vectoriser toute une gamme d'agents thérapeutiques vers le SNC de manière non invasive. Dans le cadre de cette approche, le LDLR (Low Density Lipoprotein Receptor), exprimé à la surface de la BHE, est une cible particulièrement intéressante. L'objectif de ce travail est le développement de nouveaux ligands du LDLR en tant que vecteurs potentiels de la BHE. Le criblage d'une librairie de peptides aléatoires dirigée contre le LDLR a permis l'identification d'un peptide 15-mer cyclique ayant une haute affinité in vitro. Une étude des relations structure/activité a ensuite été menée afin d'améliorer l'affinité pour le LDLR et d'augmenter la stabilité plasmatique de ce peptide. Cette étude a abouti à l'identification d'un nouveau peptide « lead » qui a été conjugué à des molécules actives afin d'évaluer la capacité du peptide à vectoriser un principe actif à travers la BHE après administration in vivo chez la souris. / Drug delivery to the central nervous system (CNS) is hindered by the presence of a physiological barrier, the blood-brain barrier (BBB). The brain endothelium is endowed with a series of transport systems, including receptor-mediated transcytosis. This system can also be used to transport therapeutics into the brain as a non-invasive manner. Among receptors expressed on the BBB, the low density lipoprotein receptor (LDLR) is relevant as a drug delivery system. This project is dedicated to the development of new peptide-based ligands of LDLR as potential BBB-vectors. The screening of a random peptide library directed to the LDLR led to the identification of hits such as a cyclic 15-mer peptide with high in vitro affinity. A structure/activity relationship study was then carried out in order to improve its affinity towards the LDLR and to increase its plasmatic stability. This study led to the identification of a new lead peptide which was conjugated to bioactive compounds in order to assess the ability of our peptide to shuttle a drug across the BBB following in vivo administration in mice.

Over the last several years, injectable drugs have been a growing area for the treatment of various therapeutic conditions and they are projected to comprise an even larger proportion among the drugs that will be available in the years to come. The injectable drugs are administered by various routes such as intramuscular (IM), intravenous (IV), subcutaneous (SC) and others, however, the majority of these drugs are administered subcutaneously. Even though subcutaneous delivery has been utilized for a number of years, very little is known about the processes governing the absorption of macromolecules from the interstitial space; and the resulting impact of these processes on the bioavailability (BA) and pharmacokinetic (PK) profiles. Also, there is no established In vitro - In vivo correlation (IVIVC) for subcutaneously administered immediate release (IR) peptide based drugs in a biorelevant manner. The contribution of IVIVC in drug development of orally administered drugs is very well known. For oral drugs, the in vivo process of drug absorption is often rate limited by the rate at which drug dissolves in the gastrointestinal tract. This can be simulated by measuring the rate of dissolution in an in vitro apparatus, which can be correlated with the in vivo absorption rate to produce an IVIVC. This research program involved efforts to develop biorelevant IVIVC methods and model for subcutaneously administered peptide based drugs. The in vivo component of this Program involves the use of clinical data from a bioequivalence (BE) study of Iplex™ [(IGF-I (Insulin like growth factor-I)/IGFBP-3 (Insulin like growth factor binding protein-3)], administered subcutaneously, that was conducted at the Center for Drug Studies (CDS), VCU School of Pharmacy in the year 2005 (Barr et. al., 2005). The PK parameters for Increlex™ (IGF-I) are calculated from the clinical data obtained from another study (Rabkin et. al., 1996). Literature research and molecular modeling research formed the basis of our hypotheses that unbound and bound IGF-I are absorbed from the blood capillaries and lymphatic capillaries respectively and that simulation of these physiologic variables is possible with the use of the modified Hanson Microette® device. The Hanson Microette® device is a vertical diffusion cell system that has been modified to simulate the pores in the capillaries with the use of a synthetic membrane. The flow and composition of circulatory fluid was simulated by the use of modified Hanks balanced salts solution (HBSS). A validated RP-HPLC (reversed-phase high performance liquid chromatography) method has been used for the analysis of IGF-1/IGFBP-3 in the in vitro samples. The in vitro permeation/release results gave the in vitro component to conduct IVIVC analysis. The General Electric (GE) healthcare sourced polycarbonate nucleopore track etched membranes were the only set of membranes that resulted in significant permeation in the in vitro experiments. IVIVC results demonstrated high inter and intra-membrane variability for the membranes (available from today’s technology) that were used to simulate the in vivo membrane characteristics. Currently, there are no validated biorelevant IVIVC methods for SC formulations. The methods described here are the basis for future in vitro method development that will be of significant value for (a) predicting the in vivo performance of SC formulations based on the in vitro data, and (b) provide a reproducible in vitro method as the basis of developing an IVIVC for other subcutaneously administered drugs. This will provide an important tool for both development and regulation of this growing class of drugs.

Doctor of Philosophy / Department of Chemistry / Duy H. Hua / Three research projects are described in this dissertation, and they are: (i) discovery of piperidine derivatives as T-type calcium channel inhibitors for the treatment of epilepsy and neuropathic pain and as protein disulfide isomerase inhibitors for the treatment of influenza viral infection; (ii) discovery of peptide-based calcitonin gene-related peptide receptor antagonists for the treatment of inflammatory pain; and (iii) synthesis of substituted 6-(dimethylamino)-2-phenylisoindolin-1-ones for the inhibition of luciferase. T-type calcium channels are important regulators of nervous system, and upregulated T-type calcium channel activities have been found to link to various types of neurological disorders, such as epilepsy and neuropathic pain. To discover novel T-type calcium channel blockers, a series of 1,4-disubstituted piperidine derivatives were designed and synthesized. Among them, compound 1-4 was found to be a good T-type calcium channel inhibitor with an IC₅₀ of 1 nM for Ca[subscript v]3.2 inhibition. It also showed 86% suppression of seizure induced death in mice and good in vivo analgesic effects on both thermal and mechanical pain thresholds in Spared Nerve Injury rat models. Therefore 1-4 can potentially be used as a T-type calcium channel blocker in the treatment of epilepsy and neuropathic pain. Influenza is a respiratory viral infection. Since viruses rely on host cell proteins for their entry, survival and replication, development of drugs targeting host cell proteins has identified as an effective strategy in controlling viral infections. We synthesized a series of 1,4-disubstituted piperidine derivatives for the inhibition of protein disulfide isomerase enzyme and influenza. Among them, 1-29 was found to possess strong anti-influenza activity (EC₅₀ = 2.5 µM). This suggests the potential use of piperidine scaffold in designing anti-influenza drugs in future. Calcitonin gene-related peptide (CGRP) receptor antagonism has been identified as a successful approach for the treatment of inflammatory pain. Therefore, a novel class of peptide antagonists of CGRP receptor was synthesized and screened for their binding affinities to the CGRP receptor and their analgesic effects on inflammatory-induced pain in rats. Among them, peptide 2-3 showed a higher binding affinity towards the CGRP receptor than previously reported peptide antagonists and exhibited analgesic effects up to 2 h in both Aδ and c-fiber pain tests. Therefore 2-3 indicates its potential use as a CGRP receptor antagonist in the treatment of inflammatory pain. Firefly luciferase is commonly used as a reporter in cells expressing a luciferase gene or its enzymatic activity under the control of a promoter of interest to assess its transcriptional activity. It has been found that some molecules such as molecules with carboxylic acid moiety can directly inhibit luciferase activity in cells. However, it is suggested that carboxylic acid moiety of the compounds may also be associated with side reactions in cells. Therefore, to study whether carboxylic acid moiety causes side effects, we designed two probe molecules, 3-1 and 3-2. Synthesis of probe molecule 3-2 is discussed. Synthesis of probe molecule 3-1 and further investigation of its luciferase inhibition will therefore be useful to understand the toxicity of carboxylic acid containing drugs in future.

Investigations detailed in this thesis constitute a part of the continuing programme of research undertaken in our laboratory on the riboflavin carrier protein (RCP) with partic­ular reference to identification and synthesis of neutralizing antigenic determinants, design of relevant epitope mimetics with improved immunogenic characteristics and relationship between their secondary structures and immunological properties. The riboflavin carrier protein is elaborated as a reproductive stratagem to ensure ade­quate vitamin deposition in the developing oocyte in the chickens. The protein is scrupu­lously conserved through evolution in terms of physico chemical and immunological char­acteristics from fish through birds to mammals, including primates. In rodents and sub­human primates immunization with the heteroantigen viz., chicken egg white RCP leads to functional neutralization of the endogenous maternal protein resulting in curtailment of early pregnancy. Thus, the crucial role of RCP in maintenance of pregnancy is established and the protein identified as a potential candidate vaccine for immimocontraception. Fur­ther studies with the reduced and carboxymethylated (RCM) RCP as the immunogen re­veal that antibodies induced by RCM-RCP are equally effective in bioneutralization of the endogenous protein. So it can be surmised that the native folded structure of RCP is not obligatory for eliciting bioneutralizing antibodies. In an attempt to identify functionally relevant regions of the protein, a panel of monoclonal antibodies (MAbs) have been raised and characterized. One of the MAbs viz., 6J32Ci2 could bring about early fetal resorp-tion when injected to mice with confirmed pregnancies. These results prompted a detail molecular immunological approach to understand underlying mechanisms. The principal aims of the present investigations include: (1) identification of neutralizing epitopes; (2) synthesis of peptidyl sequences incorporating these determinants; (3) an understanding of the structure, antigenic and immunogenic characteristics of these peptides; (4) correlation of conformational and antigenic characteristics; (5) rational design and synthesis of peptide analogs with greater propensity to assume predicted secondary structures; (6) analysis of conformation dependency of peptide antigens and the importance of such conformation in generating an optimal B-cell response; (7) the efficacy of the antibodies elicited by these Peptide antigens in neutralizing endogenous protein with the ultimate aim of designing synthetic vaccines. Chapter 1 of this thesis deals with a general introduction summarizing the current status of knowledge regarding the chemistry and biology of RCP as well as synthetic pep­tides as potential immunogens. Chapter 2 outlines details of the experimental procedures adopted. Chapter 3 describes the results of investigations on the C-terminal fragment (residues 200-219) of cRCR The main consideration in selecting this sequence for the design of a potential peptide-based vaccine relied on the epitopic specificity of the neu­tralizing MAb 6S2C12. Epitope mapping using the Pepscan method revealed that the monoclonal antibody recognizes a core sequence corresponding to residues 203-210 of the cRCP. A 21-residue synthetic peptide (C-21) comprising this epitope was synthesized and antibodies elicited to the peptide conjugated to two different carriers, namely diphtheria toxoid and purified protein derivative (PPD) for T-cell help. In both active and passive immunoneutralization experiments, the peptide specific neutralizing antibodies interfered with the biological function of the protein and hence either protected from pregnancy or caused early fetal resorption in rodents as well as in sub-human primates. The conforma-tional properties of the peptide in aqueous buffers were analyzed from circular dichroism which revealed the absence of any ordered structure in the native C-21 peptide. Theoreti­cal predictions of secondary structure suggested a propensity for an t*-helical structure for this fragment in the native protein. Therefore, influence of the helix-promoting solvent, vizM 2,2,2,trifluroethanol (TFE) on the C-21 peptide was investigated. Addition of TFE resulted in spectral changes with negative bands at 208 and 222 nm and a positive band at 190 rim which are typical of an a-helix. To gain more information on the conformational characteristics of this peptide, it was considered worthwhile to stabilize the native peptide in an a-helical conformation based on simple rational design principles. Towards this end, four analogs of the parent peptide were synthesized and helix stabilization was sought to be achieved by introducing either salt bridges or back-bone conformational constraints such as by incorporating a-amino isobutyric acid at appropriate positions. In all the analogs, the core sequence, recognised by the neutralizing MAb 6B2C12 was maintained intact to ensure induction of antibodies capable of recognizing the native protein. CD spectral analysis of the analog peptides indicated that all the engineered peptides had varying degrees of enhanced helicities as compared to the parent peptide. The immunogenicity of each analog was studied by to the relevant peptide-diphtheria toxoid conjugates and analyzing their reactivities with the native protein by direct and competitive ELISA. The results revealed that these engi­neered conformational analogs axe highly immunogenic eliciting high titers of anti-protein antibodies. The relative affinities of these antibodies to bind cRCP were investigated. The antibodies to peptide analogs had higher affinities for the native protein and a positive correlation was found between the helical content of the peptide antigen in question and the relative affinity of corresponding antibody. The antibodies directed to all the peptide analogs could block the function of RCP resulting in early embryonic resorption when ad­ministered to pregnant mice. An interesting pattern of immunological cross-reactivity has been observed with the native and designed peptides. Antibodies raised to constrained helical analogs could bind the C-21 peptide which is structurally flexible. In contrast, the antibodies raised to the flexible native peptide antigen were inefficient in recognizing the structured peptides. The ability of all the peptide antibody to bind the native protein has been interpreted in terms of a conformationally flexible C-terminus region in cRCP. Chapter 4 details investigations on a 21-residue peptide (N- 21) from the N-terminiis (4-24) of the protein. Selection of this peptidyl sequence relied on theoretical prediction of potential sequential determinants on RCP other than at C-terminus as well as on the outcome of immunoneutralisation experiments using antibodies to egg yolk RCP which lacks the relevant C-terminal determinants. The structure of this peptide in solution was analyzed by two dimensional NMR and CD. NMR experiments revealed the presence of two structured regions in the peptide. Diagnostic nuclear Overhauser effects characteristics of reverse turns or short frayed helical segments over residues 3-9 and 18-21 of the peptide were obtained. CD spectra showed the presence of a strong, negative band at 204 nm over a wide range of solvent conditions, a feature which has been interpreted in terms of a "polyproline Il-like" segment encompassing residues 11-16 which corresponds to an interesting (X-Pro)^ repeat in the N-21 sequence. Specific antibodies were generated to this peptide as a conjugate with diphtheria tox­oid. Administration of the antipeptide antibodies could neutralize the protein in vivo as demonstrated by early embryonic loss in pregnant mice. In limited experiments the anti­peptide antibodies showed propensity to protect bonnet monkeys from pregnancy over a few consecutive ovulatory cycles when titres are maintained elevated by periodic boosting. To address the relationship between peptide structures and antigenicity, epitope mapping of this antipeptide antibodies as well- as the polyclonal antibodies to native RCP was undertaken using the Pepscan method. The results reveal that antigenic regions correspond well to conformationally well-defined elements of structure with the polyproline II-like seg­ment being a common antigenic determinant on both the peptide and the native protein. These observations are suggestive of the involvement of both the N and C-terminal regions of RCP in terms of its binding to putative plasma membrane receptors.

The present work demonstrates how a peptide-based material can be obtained for the biosorptive recovery of metals from contaminated industrial wastewater. Starting with Phage surface display for the initial identification and optimization of gallium-binding peptides, all the following application-focussed experiments are based on chemically synthesized peptides. Two chromatography-based biopanning methods for the identification of gallium-binding peptides from a commercial phage display library were developed. Five gallium-binding peptide sequences were identified and evaluated to show good gallium-binding properties. Furthermore, the biosorption of free gallium and arsenic by gallium-binding bacteriophage clones was investigated. A large influence of the pH-value on the respective interactions was demonstrated. Mutagenesis experiments were also carried out for a bacteriophage clone expressed peptide, in which a cysteine pair systematically replaced amino acids. Biosorption experiments with the resulting seven different bacteriophage mutants suggested a relationship between the rigidity of the peptide structure and the gallium-binding properties. In isothermal titration experiments, the thermodynamics of the interaction between gallium and the peptides as chemically synthesized derivatives were characterized, independent of the bacteriophage. The peptides differed strongly in their interaction with gallium, and in some cases, the complex formation with gallium depended strongly on the surrounding buffer conditions. The peptide with the amino acid sequence NYLPHQSSSPSR has particularly promising gallium-binding properties. Computer modeling suggests the probable structure of the peptide in aqueous solution and postulates a possible binding site for gallium. The side-selective and covalent immobilization of the peptides on a polystyrene matrix led to the creation of a biocomposite for the biosorptive recovery of gallium. The sorption performance and desorbability of the peptide-based biosorption materials were determined in studies with model solutions and real waters from the semiconductor industry.  :EIDESSTATTLICHE VERSICHERUNG II SUMMARY 7 CHAPTER I. 8 Utility of biotechnological approaches in resource technology 9 Phage Surface Display for the recovery of inorganic binding peptides 14 Gallium – Example of a high-tech metal 22 Aims and context of the present work 22 CHAPTER II. 25 Author contributions 25 Abstract 25 Introduction 26 Materials and Methods 28 2.1 Phage Display Library system 28 2.2 Biopanning experiments 29 2.3 Single clone identification 31 2.4 Single clone binding studies 31 Results 32 3.1 Immobilization of gallium ions 32 3.2 Biopanning experiments 33 3.3 Single clone binding studies 38 Discussion 39 4.1 Gallium ions as biopanning target 39 4.2 Phage clone selection 40 Conclusion 44 Acknowledgements 45 CHAPTER III. 46 Author contributions 46 Abstract 47 Introduction 47 Materials and Methods 49 2.1 Handling of phage display library clones 49 2.2 Site-directed mutagenesis experiments 50 2.3 Biosorption experiments 51 Results and Discussion 52 3.1 Experimental context 52 3.2 Original phage clone characterization 53 3.3 Site-directed mutagenesis experiments 56 3.4 Mutant phage clone characterization 57 Conclusions 59 Acknowledgements 60 CHAPTER IV. 61 Author contributions 61 Textual and graphical abstract 62 Introduction 63 Methods 65 2.1 Peptides 65 2.2 Isothermal titration microcalorimetry (ITC) 65 2.3 Preparation of peptide conjugates 65 2.4 Biosorption studies 66 2.5 Model calculation of peptide C3.8 67 Results and Discussion 68 3.1 Interaction studies of free peptides in solution 68 3.2 Biosorption studies with peptide polystyrene conjugates 71 3.2.1 Covalent and site-selective immobilization of peptides 71 3.2.2 Interaction of peptide conjugates with gallium 72 3.2.3 Interaction of peptide conjugates with arsenic 73 3.2.4 Continuous experiments 73 3.3 Model calculation for peptide C3.15 75 Counclusion 76 Acknowledgment 77 CHAPTER V. 78 Obtained insights for the selection of metal-binding peptides in biopanning experiments 79 Conclusions for the development of peptide-based materials for the biosorptive recovery of metal ions from aqueous solutions 81 REFERENCES 85 APPENDIX 94 SUPPORTING INFORMATION FOR CHAPTER IV 94 LIST OF FIGURES 99 LIST OF TABLES 100 LIST OF ABBREVIATIONS 101 LIST OF CHEMICALS 104 ACKNOWLEDGEMENTS 106 CURRICULUM VITAE 109 LIST OF PUBLICATIONS 111

No description available.