Understanding the cellular membrane interaction with membrane active biomolecules and antimicrobial agents provides an insight in their working mechanism. Here, we studied the effect of antimicrobial agents; a recently developed peptidomimetics E107-3 and colistin as well as the N-terminal helix H0, of Endophilin A1 on the lipid bilayer. It is important to discern the interaction mechanism of antimicrobial peptides with lipid membranes in battling multidrug resistant bacterial pathogens. We study the modification of structural and mechanical properties with a recently reported peptidomimetic on lipid bilayer. The compound referred to as E107-3 is synthesized based on the acylated reduced amide scaffold and has been shown to exhibit good antimicrobial potency. This compound increases lipid bilayer permeability as indicated by our vesicle leakage essay. Micropipette aspiration experiment shows that exposure of GUV to the compound causes the protrusion length Lp to spontaneously increase and then decrease, followed by GUV rupture. Solution atomic force microscopy (AFM) is used to visualize lipid bilayer structural modulation within a nanoscopic regime. This compound induces nanoscopic heterogeneous structures rather than pore like structures as produced by melittin. Finally, we use AFM-based force spectroscopy to study the impact of the compound on lipid bilayer’s mechanical properties. With the incremental addition of this compound, we found the bilayer puncture force decreases moderately and a 39% decrease of the bilayer area compressibility modulus KA. To explain our experimental data, we propose a membrane interaction model encompassing disruption of lipid chain packing and extraction of lipid molecules. The later action mode is supported by our observation of a double-bilayer structure in the presence of fusogenic calcium ions. Polyanionic Lipopolysaccharides LPS are important in regulating the permeability of outer membrane (OM) of gram-negative bacteria. To initiate the bactericidal activity of polymyxins, it is essential to impair the LPS-enriched OM. Here, we study the mechanism of membrane permeability caused by colistin (Polymyxin E) of LPS/phospholipid bilayers. Our vesicle leakage experiment showed that colistin binding enhanced bilayer permeability; the maximum increase in the bilayer permeability was positively correlated with the LPS fraction. Addition of magnesium ions abolished the effect of LPS in enhancing bilayer permeabilization. Solution atomic force microscopy (AFM) measurements on planar lipid bilayers shows the formation of nano- and macro clusters which protruded from the bilayer by ~2nm. Moreover, increasing the fraction of LPS or colistin enhances the formation of clusters but inhibits by magnesium ions addition. To explain our experimental data, we proposed a lipid-clustering model where colistin binds to LPS to form large-scale complexes segregated from zwitterionic phospholipids. The discontinuity (and thickness mismatch) at the edge of LPS-colistin clusters will create a passage that allows solutes to permeate through. The proposed model is consistent with all data obtained from our leakage and AFM experiments. Our results of LPS-dependent membrane restructuring provided useful insights into the mechanism that could be used by polymyxins in impairing the permeability barrier of the OM of Gram-negative bacteria. Also, we studied the effect of helix H0 of a membrane modification inducing protein endophilin, on planar bilayer. We obtained transmembrane defects on the bilayer when scanned.with AFM.

Des peptides antigéniques sont présentés à la surface des cellules cancéreuses ou infectées par un virus pour être reconnus par des cellules du système immunitaire. Cette reconnaissance déclenche alors une réponse immunitaire spécifique contre les cellules présentatrices ciblées. L'objectif de ce projet est de stimuler cette réponse immunitaire afin qu'elle soit un moyen thérapeutique pour détruire spécifiquement les cellules cancéreuses ou infectées. Toutefois, les peptides antigéniques ne peuvent pas être administrés tels quels, du fait de leur pauvre stabilité en milieu biologique. Il est donc nécessaire de les modifier afin qu’ils soient plus résistants. Le défi de ce projet est de synthétiser des peptidomimes bio-résistants, capables de reproduire la même réponse immunitaire que celle du peptide antigénique naturel. / Abstract

Hypoxia inducible factor (HIF)-1α is a heterodimerically-activated transcription factor central to the cellular response to hypoxic environments and is often upregulated in cancer. Binding of HIF-1α to the co-activator p300 is necessary for the hypoxia-induced transcription of many oncogenic proteins. The aim of this project was to develop novel small molecule inhibitors of the HIF-1α/p300 protein-protein interaction (PPI). Initial work focused on designing, validating and optimising two high-throughput competition binding assays to screen for inhibitors of the PPI (Chapter 2). Alongside these, zinc ejector assays for both p300 and KDM4A proteins were developed to probe the mechanism of action and selectivity. Analysis of hits from a natural product high-throughput screen (HTS) revealed two compound classes; benzoquinones and 2-substituted indandiones, which modulate the PPI. The potency of these series correlated with the reactivity of the core functional groups, which act as electrophiles to covalently modify reactive cysteines, ejecting structural zinc and disrupting the p300/KDM4A protein fold (Chapter 3). Conjugating electrophilic groups to putative HIF-1α/p300 inhibitors did not replicate the activity of the zinc ejecting HTS hits (Chapter 4). Further work focused on non-covalent inhibitors of the HIF-1α/p300 interaction, first with peptide truncates, and then rationally designed α-helix peptidomimetics. An 11mer truncate showed encouraging activity (IC50 ≈ 70 μM), and corresponded to a key α-helix in the HIF-1α C-terminal transactivation domain. Three distinct double-sided scaffolds were used to imitate up to five hot-spot ampiphilic residues on this α-helix (Chapter 6 and 7). Of the 35 compounds screened, only modest inhibition was observed (IC50 ≈ 200-500 μM). Future work will look to conjugate electrophilic functionality onto the 11mer peptide in an attempt to gain potency from zinc ejection, while maintaining selectivity for p300.

A series of novel alpha-aminoxy peptides have been recently developed, and showed a therapeutic potential for the treatment of CI- channel dysfunctional diseases. The present study aimed to investigate the pharmacokinetics of five structural related tx-aminoxy peptides (P1 to P5), and effect of structural modifications on their pharmacokinetic properties. / P1 showed significantly low intestinal permeability due to P-gp-mediated efflux. Structural modifications resulted in alterations of transport mechanisms from P-gp-mediation (P1, P2) to multidrug resistance-associated protein (MRP)-mediation (P3), MRP plus breast cancer resistance protein (BCRP)-mediation (P4) active transport, or even passive paracellular diffusion (P5) without any efflux transporters involved. Comparing with P1, the absorbable permeability in Caco-2 monolayer increased to about 7-fold (P3), 4-fold (P4) and 11-fold (P5), respectively, and the absorption through intestine in SPIP model significantly increased to about 36-fold (P2), 42-fold (P3), 55-fold (P4) and 102-fold (P5), respectively. P1 was unstable in the GI tract with 41% degradation in SGF within 1 h and 47% degradation in SIF within 3 h. The other four peptides were much more stable than P1 with degradation less than 6% under the same incubated conditions. For the hepatic metabolism, about 31% of P1 was metabolized by rat liver S9 within 30 min, while the metabolic stability was significantly improved to 3.3-fold (P3), 2.9-fold (P4) and 7.5-fold (P5), respectively with no metabolism for P2. Their metabolism was mainly via oxidation catalyzed by CYP enzymes to form hydroxylated metabolites. After i.v. administration (5 mg/kg), both P1 and P5 was eliminated rapidly. P1 mainly distributed to liver and lung, while P5 to kidney and intestine. P1 was cleared mainly through metabolism via oxidation followed by sulphation (∼80% of the dose), while P5 was mainly eliminated as an intact form (∼53% of the dose). Oral bioavailability of P1 was low (0.36%) due to instability in the GI tract and poor intestinal absorption mediated by P-gp efflux transport. Oral bioavailability of P5 was improved to about 3-fold comparing with P1 but still low mainly because of poor intestinal absorption through passive diffusion and some unknown factor(s). / The present studies demonstrated that our rationale for the structural modifications of the designed cc-aminoxy peptides is an effective way to improve their intestinal absorption, gastrointestinal and metabolic stability, and appropriate pharmacokinetic properties. Our findings also provide scientific evidence to support further development of better alpha-aminoxy peptide cadidates with high oral bioavailability and appropriate pharmacokinetic properties. / Three absorption models, including Caco-2 cell monolayer, Ussing chamber and in situ rat single-pass intestinal perfusion (SPIP) model, were used. The stability in gastrointestinal (GI) tract was determined using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The hepatic metabolism was investigated in rat liver subcellular fractions and human liver microsome. P1 and P5 were selected for pharmacokinetic study in rats. / Ma, Bin. / Adviser: Ge Lin. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 199-215). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

3C-like protease (3CLpro) of severe acute respiratory syndrome-coronavirus (SARS-CoV) is required for autoprocessing of the polyproteins 1a and 1ab, and is a potential target for treating coronaviral infection. To obtain a thorough understanding of its substrate preference, we created a substrate library of 19 x 8 variants by performing saturation mutagenesis on the autocleavage sequence at P5 to P3' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer (FRET). The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high beta-sheet propensity P4 prefers small hydrophobic residues: P2 prefers hydrophobic residues without beta-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1' position prefers small residues, while P2' and P3' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence. / Inhibition of SARS-CoV 3CLpro proteolytic activity suppresses virion replication and virus-induced cytopathic effects. Peptidomimetic inhibitors with nitrile warheads, which inhibit Cys protease activity, have been applied for clinical therapy. To investigate whether the nitrile group can target 3CLpro, a series of nitrile-based peptidomimetic inhibitors with various protective groups, peptide length and peptide sequences were synthesized. Inhibitor potency in terms of IC50 and Ki values was determined by FRET assay. Most of these nitrile-based inhibitors in micromolar range can significantly reduce 3CLpro activity. The most potent inhibitor is the tetrapeptidomimetie inhibitor linked with carbobenzyloxy (cbz) group 'cbz-AVLQ-CN' with IC50 and Ki values of 5.9 +/- 0.6 muM and 0.62 +/- 0.11 muM respectively. Crystal structures of 3CLpro-inhibitor complexes demonstrated that nitrite warhead covalently bonded to Cys145, while P1 -- P4 residues interacted with 3CLpro as substrate bound. The cbz group in 'cbz-AVLQ-CN' flipped into a cavity of Gu166 -- Pro168, providing an extra binding force to enhance inhibitor potency. In conclusion, the nitrile-based peptidomimetic inhibitor with cbz group is a convincing model for drug development. / Substrate specificities of various 3CLpro were further investigated by using the substrate library of SARS-CoV 3CLpro. Among various viral strains, the proteases of HCoV-NL63, HCoV-OC43 and infectious bronchitis virus (IBV) were selected from group I, IIa and III respectively for specificity profiling. Their proteolytic rates against 19 x 8 variants were obtained by FRET assay, and correlated with structural properties of substituting residues. Like SARS-CoV 3CLpro in group IIb, these 3CLpro consistently prefer small hydrophobic P4 residues, positively charged P3 residues, hydrophobic P2 residues without beta-branch, P1-Gln and small P1' residues. These proteases also tend to accommodate P5 and P3' residues with positive charge, and P2' residues with small size. In contrast, their preferences on secondary structure are diverse. Correlation was found between IBV 3Clpro activity and beta-sheet propensity at P5 position, while no strong correlation with secondary structure propensities was observed in HCoV-NL63 and HCoV-0C43. Collectively, all 3CLpro share universal preferences on charge, side chain volume and hydrophobicity, but not secondary structure. Their relative activities against universal and specific super-active substrates were elevated to 1.4 -- 4.3, showing synergetic effects by combining preferred residues. These substrates were examined by group I HCoV-229E and group IIa HCoV-HKU1 in parallel. Their activities were highly comparable to those of other group members. / Chuck, Chi Pang. / Adviser: Chi-Cheong Wan. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves [179]-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Proteins can be described as the “machineries” responsible for almost all tasks in the levels of organizational complexity in multi-cellular organisms namely: the cells, tissues, organs and systems. Any disorder in the function of a protein at any of these levels could result in disease, and a study of protein function is critical to understanding the pathological features of the disease at the molecular level. A quick glance at these abundantly present proteins reveals two striking features: large diversity of biological function, and the variations in structural complexity, which varies from simple random coils, to turns and helices, and on to structured assemblies of turns, helices and sheets. Over the past few years, more research efforts have been channeled to the application of synthetic research to protein dynamics, most especially in disease conditions. This provides insight into the design and development of chemical tools capable of modulating protein functions .Some of such tools include small molecules, peptides and peptidomimetics. In this work, we have described the application of these tools to three (3) different disease systems topping the list of incurable diseases: HIV, Diabetes, and Cancer. We have designed and developed chemical probes to facilitate a better understanding of major “culprit” proteins underlying the pathological conditions associated with these diseases. Our designed chemical probes were capable of modulating protein functions by producing the desired effects: inhibition of protein-protein interactions.

Peptidomimetics derived from the bioactive hexapeptide angiotensin IV (Ang IV, Val1-Tyr2-Ile3-His4-Pro5-Phe6) have been designed and synthesized. These peptidomimetics are aimed at inhibiting the insulin-regulated amino peptidase (IRAP), also known as the AT4 receptor. This membrane-bound zinc-metallopeptidase is currently under investigation regarding its potential as a target for cognitive enhancers. The work presented herein was based on stepwise replacement of the amino acid residues in Ang IV by natural and unnatural amino acids, non-peptidic building blocks, and also on the introduction of conformational constraints. Initially, we focused on the introduction of secondary structure mimetics and backbone mimetics. The C-terminal tripeptide His-Pro-Phe was successfully replaced by a γ-turn mimetic scaffold, 2-(aminomethyl)phenylacetic acid (AMPA), which was coupled via an amide bond to the carboxyl terminus of Val-Tyr-Ile. Substitution of Val-Tyr-Ile, Val-Tyr, Tyr-Ile and Tyr, respectively, by 4-hydroxydiphenylmethane scaffolds comprising a 1,3,5-substituted benzene ring as a central moiety unfortunately rendered peptidomimetics that were less potent than Ang IV. The subsequent approach involved the introduction of conformational constraints into Val-Tyr-Ile-AMPA by replacing Val and Ile by amino acid residues appropriate for disulfide cyclization or ring-closing metathesis. Chemically diverse structures encompassing an N-terminal 13- or 14-membered macrocyclic tripeptide and a C-terminal non-peptidic moiety were developed. Tyr2 and AMPA were modified to acquire further knowledge about the structure-activity relationships and, in addition, to improve the metabolic stability and reduce the polarity. Several of the compounds displayed a high capacity to inhibit IRAP and exhibited Ki values in the low nanomolar range. Hence, the new compounds were more than ten times more potent than the parent peptide Ang IV. Enhanced selectivity over the closely related aminopeptidase N (AP-N) was achieved, as well as improved stability against proteolysis by metallopeptidases present in the assays. However, additional investigations are required to elucidate the bioactive conformation(s) of the relatively flexible N-terminal macrocycles. The compounds presented in this thesis have provided important information on structure-activity relationships regarding the interaction of Ang IV-related pseudopeptides and peptidomimetics with IRAP. The best compounds in the series constitute important starting points for further discovery of Ang IV peptidomimetics suitable as tools in the investigation of IRAP and other potential targets for Ang IV. The literature presents strong support for the hypothesis that drug-like IRAP inhibitors would serve as a new type of future cognitive enhancers with potential use in the treatment of cognitive disorders, e.g. Alzheimer’s disease.

Inhibiting protein-protein interactions to achieve a therapeutically desired effect has been a goal in the field of drug discovery for decades. Recently, advances in peptidomimetics have led researches to the use of cyclized peptides to achieve this goal. Cyclization of linear peptides restricts the number of conformations of the peptide, increasing the peptide's affinity to binding to the desired target. Cyclization also stabilizes the peptide, allowing the peptide to be resistant to proteases. This study explores the optimization of solution phase synthesis of an important integrin-mediated cell adhesion cyclic peptide for the therapeutic inhibition of multiple myeloma, cHYD1. cHYD1 was originally synthesized via solid phase peptide synthesis, and the need for a scaled up synthesis version was needed after positive bioactivity results were obtained. Chapter 3 includes the molecular modeling exploration of a possible new mechanism to which cyclized peptides could work, in which, rather than a recognition and non-recognition strand being implemented, a specific directional face is used for protein-protein interaction. This was done with the implementation of an antagonistic cyclic peptide to replace human growth hormone in its interaction with the human growth hormone receptor, and the subsequent di-cyclic peptide agonist.

A scourge of Type I and Type II diabetes impacts the health of hundreds of millions worldwide. The number and prevalence of diabetics are expected to rise dramatically in the next two decades. Diabetes is defined by chronic hyperglycemia which can result in a number of detrimental and costly metabolic, renal, cardiovascular and neurological disorders. Identification of at risk individuals and effective blood glucose management are critical to improving diabetic outcomes and preventing hyperglycemic complications. Diabetes prevention and treatment is limited by the understanding of islet function and mass in the diabetogenic and diabetic state. The islets of Langerhans are dispersed throughout the pancreas and comprise <2% of the pancreatic mass. The reclusive nature of islet cells presents unique challenges understanding disease development. No agent capable of exclusively targeting pancreatic β-cells within the islet has been discovered and the lack of targeting agent specificity impedes efforts to: quantify β-cell mass and develop novel therapeutics. We propose β-cell targeting can be improved by targeting unique combinations of receptors simultaneously with multivalent ligands. A synthetic multivalent agent composed of two β-cell specific diabetic therapeutics, glucagon-like peptide-1 (GLP-1) and glibenclamide (Glb), targeted against the GLP-1R and the sulfonylurea-1 receptor (SUR1) is a lead compound for the development of specific bi-functional islet cell targeting agents for use in the in vivo detection and treatment of β -cells. Herein, we describe the synthesis and initial characterization of a heterobivalent ligand composed of GLP-1 coupled to Glb. The heterobivalent ligand binds to an unaltered β-cell line with increased specificity relative to a human pancreatic exocrine cell line. Additionally, receptor cross-linking modifies β-cell signaling. Exposure of β-cells to the heterobivalent ligand results in antagonism of SUR1-Ca²⁺ signaling and equipotent agonism of GLP-1R-cAMP signaling, in comparison to the cognate monomeric ligands (Glb and GLP-1). Perturbations in intracellular signaling modifies β-cell insulin secretion resulting in decreased basal insulin secretion and with maintained yet reduced ability to potentiate β-cell glucose stimulated insulin secretion. GLP-1/Glb β-cell specificity and functional modulation suggests combinatorial receptor targeting is an effective strategy for the development of bi-functional cell-specific targeting agents, warranting further investigation and optimization.

The main focus of this thesis is on photochemical modulation of molecular recognition in various host-guest systems. This involves the design, synthesis and integration of light-triggered conformational switches into peptidomimetic guests and molecular tweezer hosts. The impact of the switches on guest and host structures has been assessed by spectroscopic and computational conformational analysis. Effects of photochemical structure modulation on molecular recognition in protein-ligand and supramolecular host-guest systems are discussed. Phototriggerable peptidomimetic inhibitors of the enzyme M. tuberculosis ribonucleotide reductase (RNR) were obtained by incorporation of a stilbene based amino acid moiety into oligopeptides between 3-9 residues long (Paper I). Interstrand hydrogen bond probability in the E and Z forms of the peptidomimetics was used as a tool for predicting conformational preferences. Considerable differences in inhibitory potency for the E and Z photoisomers were demonstrated in a binding assay. In order to advance the concept of photomodulable inhibitors, synthetic routes towards amino acid derivatives based on the more rigid stiff-stilbene chromophore were developed (Paper II).  The effect of E-Z isomerization on the conformational properties of peptidomimetic inhibitors incorporating the stiff-stilbene chromophore was also assessed computationally (Paper III). It was indicated that inhibitors with the more rigid amino acid derivative should display larger conformational divergence between photoisomers than corresponding stilbene derivatives. Bisporphyrin tweezers with enediyne and stiff-stilbene spacers have been synthesized, and the conformational characteristics imposed by the spacers have been studied and compared to a glycoluril linked tweezer. The effects of spacers on tweezer binding of diamine guests and helicity induction by chiral guests have been investigated (Paper IV). Connections between spacer flexibility and host-guest binding strength have been established. The structural properties of the stiff-stilbene spaced tweezer made it particularly susceptible to helicity induction by both monotopic and bitopic chiral guests. Finally, the possibility of photochemical bite-size variation of tweezers with photoswitchable spacers has been assessed. Initial studies have shown that photoisomerization of the tweezers is possible without photochemical decomposition. Conformational analyses indicate that isomerization should impact binding characteristics of the tweezers to a significant extent (Paper V).