Academic literature on the topic 'Multivalent recognition'

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

Several biological macromolecules adopt bivalent or multivalent interactions to perform various cellular processes. In this regard, the development of molecular constructs presenting multiple ligands in a specific manner is becoming crucial for the understanding of multivalent interactions and for the detection of target macromolecules. Nucleic acids are attractive molecules to achieve this goal because they are capable of forming various, structurally well-defined 2D or 3D nanostructures and can bear multiple ligands on their structures with precisely controlled ligand–ligand distances. Thanks to the features of nucleic acids, researchers have proposed a wide range of bivalent and multivalent binding agents that strongly bind to target biomolecules; consequently, these findings have uncovered new biosensing strategies for biomolecule detection. To date, various bivalent and multivalent interactions of nucleic acid architectures have been applied to the design of biosensors with enhanced sensitivity and target accuracy. In this review, we describe not only basic biosensor designs but also recently designed biosensors operating through the bivalent and multivalent recognition of nucleic acid scaffolds. Based on these designs, strategies to transduce bi- or multivalent interaction signals into readable signals are discussed in detail, and the future prospects and challenges of the field of multivalence-based biosensors are explored.

Multivalency effects are essential in carbohydrate recognition processes as occurring on the cell surface. Thus many synthetic multivalent glycoconjugates have been developed as important tools for glycobiological research. We are expanding this collection of molecules by the introduction of carbohydrate-scaffolded divalent glycothymine derivatives that can be intramolecularily dimerized by [2 + 2] photocycloaddition. Thus, thymine functions as a control element that allows to restrict the conformational flexibility of the scaffolded sugar ligands and thus to “organize” multivalency. With this work we add a parameter to multivalency studies additional to valency.

Multivalent interactions frequently occur in biological systems and typically provide higher binding affinity and selectivity in target recognition than when only monovalent interactions are operative. Thus, taking inspiration by nature, bivalent or multivalent nucleic acid aptamers recognizing a specific biological target have been extensively studied in the last decades. Indeed, oligonucleotide-based aptamers are suitable building blocks for the development of highly efficient multivalent systems since they can be easily modified and assembled exploiting proper connecting linkers of different nature. Thus, substantial research efforts have been put in the construction of dimeric/multimeric versions of effective aptamers with various degrees of success in target binding affinity or therapeutic activity enhancement. The present review summarizes recent advances in the design and development of dimeric and multimeric DNA-based aptamers, including those forming G-quadruplex (G4) structures, recognizing different key proteins in relevant pathological processes. Most of the designed constructs have shown improved performance in terms of binding affinity or therapeutic activity as anti-inflammatory, antiviral, anticoagulant, and anticancer agents and their number is certainly bound to grow in the next future.

Developing highly active, multivalent ligands as therapeutic agents is challenging because of delivery issues, limited cell permeability, and toxicity. Here, we report intrinsically cell-penetrating multivalent ligands that target the trinucleotide repeat DNA and RNA in myotonic dystrophy type 1 (DM1), interrupting the disease progression in two ways. The oligomeric ligands are designed based on the repetitive structure of the target with recognition moieties alternating with bisamidinium groove binders to provide an amphiphilic and polycationic structure, mimicking cell-penetrating peptides. Multiple biological studies suggested the success of our multivalency strategy. The designed oligomers maintained cell permeability and exhibited no apparent toxicity both in cells and in mice at working concentrations. Furthermore, the oligomers showed important activities in DM1 cells and in a DM1 liver mouse model, reducing or eliminating prominent DM1 features. Phenotypic recovery of the climbing defect in adult DM1Drosophilawas also observed. This design strategy should be applicable to other repeat expansion diseases and more generally to DNA/RNA-targeted therapeutics.

The pentraxins, serum amyloid P component (SAP) and C-reactive protein (CRP) are target proteins for the development of treatments for amyloidosis and ischaemic injury, respectively, in humans. This study reports the first multivalent ligands capable of targeting all five SAP binding sites simultaneously. Ligands presenting five or ten D-proline headgroups and composed of five peptideglycol linkers emanating from ε-N-substituted lysine residues on a central cyclic peptide core were synthesised by solid phase peptide synthesis. The sub-nanomolar, ~250pM, binding affinity approximated by Isothermal Titration Calorimetry (ITC) for one decavalent ligand is the strongest affinity for an SAP binding ligand currently known and stronger than the affinity of SAP binding to amyloid deposits. X-ray crystallography and mass spectrometry shows the decavalent ligands noncovalently cross-linking two SAP pentamers, in the same manner observed for lead drug candidate CPHPC, but with increased affinity. In addition, the binding of SAP with N-acetyl D-proline has been investigated by x-ray crystallography. Using a 1.5Å resolution structure the exact interaction of the headgroup used in CPHPC, penta- and decavalent ligands, was investigated. The results show potential for an electrostatic interaction between the carbonyl oxygen of acetyl from the ligand and the side chain amide of Gln148, which has not previously been considered. Applications of multivalent binding are still emerging; in this study, bivalent ligand BPC8 was used as an additive to crystallise CRP from the Rat (rCRP) in non-covalently cross-linked decameric complexes. Previous x-ray crystallography studies have failed due to extreme radiation sensitivity of the crystals produced. This problem has been overcome with a complete dataset obtained from a single crystal at 3.2Å. No inter-protein contacts are seen between pentamers in the decamer complex, therefore the use of bivalent ligands has facilitated the observed crystal packing. Multivalent ligands are suggested as tools for overcoming difficult crystallisation issues.

Des supramolécules basées sur la reconnaissance de viologènes par des curcubit[n]urils (CB[n]) ont été étudiées. Les systèmes développées incluent des [3]-, [4]- et [7]pseudorotaxanes, composés de multimères à base de viologène et de CB[7] ou CB[8]. L’étude physicochimique a montré que ces supramolécules sont interconverties électrochimiquement entre un état complexé, défini par les pseudorotaxanes, et un état dissocié comprenant chacun des partenaires. Le désassemblage résulte d’une pimérisation intra/intermoléculaire entre radicaux viologènes.L’étude physicochimique de complexes pentacoordinés basés sur la reconnaissance d’azo-aryl-imidazoles par une porphyrine à anse phénanthroline a été menée; l’objectif étant d’utiliser les propriétés d’isomérisation photoinduite trans-cis d’azo-chromophores pour dissocier ces complexes. Les complexes initiaux sont alors régénérés par relaxation thermique. La stabilité et les propriétés cinétiques des complexes pentacoordinés ont été évaluées
Supramolecules based on the recognition of redox-active bipyridiniums by cucurbit[n]uril (CB[n]) have been studied. The investigated systems include a [3]-, a [4]- and a [7]pseudorotaxane, each of them composed of a multimeric viologen-based thread molecule and CB[7] or CB[8]. The physicochemical approach emphasized that these systems can be electrochemically switched between a complexed state, defined by the pseudorotaxanes, and an uncomplexed state comprising their components. The disassembly results from intra/intermolecular pimerization of the viologen radicals.A physicochemical approach of pentacoordinated complexes of a phen-strapped porphyrin with azo-arylimidazoles has been undertaken with the aim to use the photoinduced trans-cis isomerization properties of azo-chromophores to dissociate the complexes. Thermal equilibration reinstates the thermodynamically favoured complexes. The strength and the kinetic properties of these pentacoordinated species have been evaluated

Aufgrund der Fähigkeit von Langerhans Zellen, welche den C-Typ Lektin (CTL) Rezeptor Langerin exprimieren, Antigene zu internalisieren und T-Zellen zu präsentieren, wurde Langerin als attraktives Ziel für neue Immunotherapien erkannt. Langerin kann Pathogene wie z.B. Viren erkennen, die zur Erhöhung der Avidität Kohlenhydratliganden multivalent präsentieren, da die monovalenten Kohlenhydratliganden nur niedrige Affinitäten für Langerin aufweisen. Die natürlichen monovalenten Kohlenhydratliganden besitzen nur niedrige Affinitäten für Langerin. Inspiriert durch die Natur stellt Multivalenz eine Strategie zur Überwindung der schwachen CTL-Kohlenhydrat-Wechselwirkung dar. Im Gegensatz zur hochmultivalenten Präsentation von Liganden mit undefinierter Anordnung hat sich diese Arbeit zum Ziel gesetzt auch die Ökonomie der Liganden zu optimieren, indem Liganden auf einer DNA Gerüststruktur so präsentiert wurden, dass sie die Distanz zwischen den Bindungstaschen des Homotrimers Langerin wiederspiegeln. Eine Untersuchung der relevanten multivalenten Bindungsmechanismen führte zu einer Anordnung der Liganden, die sowohl statistisches Rebinding als auch den Chelate Effekt einbezog. Der Rebinding Effekt wurde als Mittel erkannt, dass nicht nur die Avidität des Liganden an einer Bindungstasche erhöht, sondern auch ausgenutzt werden kann, um den Chelate Effekt zu amplifizieren. Diese Methode stellt eine Möglichkeit dar niedrige oder nicht vorhandene Multivalenzeffekte bei der bivalenten Präsentation von Liganden zu überwinden, wenn hochaffine Liganden nicht zur Verfügung stehen. Eine Kombination dieser Strategie mit der Entwicklung eines neuen selektiven Liganden für Langerin führte zu dem stärksten bekannten Langerinbinder (IC50 = 300 nM). Die Ligand-PNA-DNA Konstrukte wurden selektiv von Langerin exprimierenden Zellen bei nanomolaren Konzentrationen internalisiert und stellen ein System dar, welches in Zukunft für den Transport von Beladungen Anwendung finden könnte.
Targeting the C-type lectin (CTL) langerin has received increasing attention as a novel immunotherapy strategy due to the capacity of Langerhans cells, which express langerin, to endocytose and cross-present antigens to T-cells. Langerin recognizes pathogens such as viruses, which present carbohydrates in a multivalent fashion to increase avidity as the monovalent carbohydrate ligands only display low affinity for langerin. Inspired by nature, multivalency has therefore been a key tool for overcoming the low affinities of CTL-carbohydrate interactions. In contrast to highly multivalent ligand presentation with undefined arrangements this work strove to optimize ligand economy by designing bivalent ligands that take the distance between the binding sites of the homotrimeric langerin into consideration by precise arrangement of ligands on DNA-based scaffolds. Studying the multivalent mechanisms at work led us to the design of ligands that take both statistical rebinding and the chelate effect into account. The rebinding effect was recognized as a tool that not only increases ligand avidity at a single binding site but in addition can be exploited to amplify the chelate effect. This method provides a solution for overcoming the low or non-existing multivalency effects when bivalently presenting low affinity ligands on a rigid scaffold if high affinity ligands are unavailable. A combination of this arrangement strategy with the development of a first langerin selective glycomimetic ligand led to the most potent molecularly defined langerin binder to date (IC50 = 300 nM). The ligand-PNA-DNA constructs were selectively internalized by langerin expressing cells at nanomolar concentrations and constitute a delivery platform for the future transport of cargo to Langerhans cells.

2009/2010
Water-soluble gold nanoparticles represent an appealing scaffold for the preparation of robust and biocompatible bioconjugates. Indeed, many examples of gold nanoparticles-bioconjugates as new materials in several fields as material science, biology and medicine have been reported in the literature. The organic monolayer protecting the metallic core plays a key role in determining the properties of the system as stability, solubility, and specific interactions with biological environment. The present thesis is focused on the functionalization of water-soluble gold nanoparticles in order to develop new tools in diagnostics, drug-delivery and enhanced immuno-sensing. Gold nanoparticles protected by mixtures of ligands of different nature have been taken into consideration in the development of the three main projects of this thesis. The first project is about the synthesis of gold nanoparticles with a gold core of 1.7 nm suited for crystallization, in order to perform diffractometric analysis aimed to solve the structure of larger systems than that already reported and to find other geometries of the gold core. To this aim, gold nanoparticles protected by a monolayer of p-mercaptobenzoic acid have been synthesized, purified and characterized. The choice of an aromatic ligand with a carboxylic group imparts stability to the clusters and plays a strategic role in crystals formation. Crystallization trials under a variety of different conditions and preliminary observations about the stability of the nanoparticles are reported. Up to now suitable crystals for X-ray analysis could not be obtained. The second project is part of an ongoing investigation of the morphological organization of the monolayer protecting gold nanoparticles in order to complete previous studies carried out in our research group. Recent results from our laboratories, obtained by ESR measurements, support the formation of “patches” domains in the mixed-monolayer of water-soluble gold nanoparticles when mixtures of perfluoroalkyl- and alkylthiolates are used to form the monolayer. The complexity of these systems may also be increased introducing functional thiolates in the monolayer in a controlled topology. The preliminary results obtained so far should be completed with other investigations using different methodologies and supported by studies also on flat surfaces. Moreover, to understand the ability of the amphiphilic thiols to phase-segregate, we thought to study also micellar aggregates. The final goal is to use this phase-segregated monolayers to create clusters of functional thiols for multivalent recognition. Water-soluble gold nanoparticles coated by amphiphilic thiols of different lipophobicity have been prepared and characterized, and new ligands suited for the studies on micelles and on 2D self-assembled monolayers have been designed and synthesized. The results of Electron Spin Resonance (ESR), Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM) studies on these systems are reported and discussed. The third project is aimed to find new synthetic strategies to obtain biocompatible gold nanoparticles presenting multiple bioactive residues for multivalent recognition processes. In particular, a mimetic of the antigen GM3 Ganglioside Lactone with demonstrated antimelanoma reactivity was introduced in the monolayer of water-soluble gold nanoparticles for the development of a biological therapy against cancer. The preparation of nanoparticles of different size and loading of the antigen-mimetic is reported, together with their characterization and the preliminary biological investigations.
Nanoparticelle di oro solubili in mezzi acquosi rappresentano una piattaforma ideale per la sintesi di bioconiugati stabili e compatibili con le cellule. Infatti la letteratura scientifica riporta molti esempi di nanoparticelle di oro coniugate con biomolecole come prototipo di nuovi materiali applicabili in diversi ambiti tra cui la scienza dei materiali, la biologia e la medicina. Il monostrato organico che protegge il nocciolo metallico riveste un ruolo fondamentale nel determinare le proprietà dell’intero sistema quali la stabilità, la solubilità e le eventuali interazioni specifiche con i sistemi biologici. La presente tesi si focalizza sulla funzionalizzazione del monostrato di nanoparticelle di oro idrosolubili finalizzata allo sviluppo di nuovi strumenti ad uso diagnostico, terapeutico, e con applicazione nel sensing immunologico. Nello sviluppo dei tre progetti principali in cui la tesi si articola, sono state studiate nanoparticelle di oro protette da miscele di ligandi di natura diversa. Il primo progetto riguarda la sintesi di nanoparticelle di oro aventi diametro del gold core di 1.7 nm adatte alla cristallizzazione, al fine di effettuare un’analisi diffrattometrica che consentisse di risolvere la struttura di nanoparticelle di dimensioni maggiori rispetto a quelle riportate in letteratura e di individuare caratteristiche strutturali quali ad esempio la geometria del nocciolo di oro. A questo scopo sono state preparate nanoparticelle di oro protette da un monostrato composto da molecole di acido p-mercaptobenzoico, che sono state purificate e caratterizzate. La scelta di un ligando aromatico avente gruppi funzionali carbossilici conferisce particolare stabilità ai clusters e riveste un ruolo strategico nella formazione dei cristalli. Le prove di cristallizzazione in diverse condizioni sperimentali e alcune osservazioni preliminari riguardo la stabilità di queste nanoparticelle sono di seguito riportate. Finora non è stato ancora possibile ottenere cristalli adatti per le analisi diffrattometriche. Il secondo progetto è parte di un’indagine rispetto alla morfologia del monostrato protettivo delle nanoparticelle di oro, finalizzata a completare studi già avviati nel nostro gruppo di ricerca. Recenti risultati ottenuti nei nostri laboratori mediante misure ESR sono fortemente indicativi relativamente alla formazione di domini a “macchie” nel monostrato di nanoparticelle idrosolubili composto da miscele di tiolati alchilici e perfluoro-alchilici. Questi sistemi possono raggiungere un elevato livello di complessità mediante l’introduzione con controllo topologico di tiolati funzionalizzati. Il completamento dei risultati preliminari mediante l’impiego di ulteriori tecniche e il supporto mediante studi su superfici piane è un obiettivo di primaria importanza. Inoltre, la comprensione del fenomeno di segregazione tra tioli anfifilici potrebbe essere agevolata da studi su sistemi di tipo micellare. L’obiettivo finale è l’applicazione di suddetta segregazione di fase nella realizzazione di clusters con monostrati recanti tioli funzionalizzati per il riconoscimento multivalente. Sono state preparate e caratterizzate nanoparticelle di oro idrosolubili protette da tioli anfifilici aventi diversa lipofobicità, e sono stati progettati e sintetizzati nuovi ligandi adatti allo studio su aggregati di tipo micellare e su monostrati bi-dimensionali. I risultati ottenuti mediante Risonanza di Spin Elettronico (ESR), Microscopia a Scansione per effetto Tunnel (STM) e Microscopia a Forza Atomica (AFM) su questi sistemi sono di seguito riportati e discussi. Il terzo progetto è finalizzato alla realizzazione di nanoparticelle biocompatibili coniugate a molteplici unità di composti farmacologicamente attivi per il riconoscimento multivalente. In particolare, un mimetico dell’antigene GM3 Ganglioside Lattone con testata attività antitumorale nei confronti di cellule di melanoma è stato introdotto nel monostrato di nanoparticelle di oro idrosolubili nello sviluppo di una terapia antitumorale di tipo biologico. La sintesi di nanoparticelle di varie dimensioni e con diversa composizione del monostrato organico recanti il mimetico di antigene, ed i risultati ottenuti dai primi test biologici sono qui di seguito riportati.
XXIII Ciclo
1983

En esta tesis doctoral el concepto de multivalencia en el reconocimiento de proteínas (lectinas) con azúcares se combinó con la idea de la química dinámica combinatoria. Esto se aplicó, no sólo para sacar ventaja del efecto de la mejor afinidad de tales sistemas multivalentes, sino también para dotar al sistema con una mayor variedad de constituciones y geometrías. La determinación de las afinidades relativas de los miembros de la biblioteca dinámica dio una visión de los requisitos necesarios para la unión entre azúcares – lectina. El primer enfoque para acceder a los sistemas multivalentes para el reconocimiento de lectinas (presentado en el capítulo 2 de la tesis), está basado en estrategias bien conocidas. Enlaces covalentes reversibles son usados para acceder a bibliotecas dinámicas combinatorias (DCLs). En esta parte del trabajo se confirmó la viabilidad del procedimiento analítico elegido. Especies diméricas, similares a las que se había conocido desde experimentos de otros grupos, mostraban buena analisabilidad de los DCLs formados. El método analítico elegido (HPLC MS) permitió la detección de las afinidades y selectividades relativas de tales constituyentes de la respectiva biblioteca. Para la elaboración de las bibliotecas dinámicas, varios intentos fueron realizados basados en el mismo concepto: Una subunidad central con múltiples puntos de conexión para favorecer interacciones reversibles. Formación de una librería dinámica basada en un conector central. Específicamente, se evaluaron los puentes di sulfuro y la formación de imina. Algunos de estos estudios resultaron ser complicados por problemas secundarios, tales como solubilidad en agua y las interacciones secundarias no deseadas de unidades centrales, debido principalmente a reacciones intramoleculares. Sin embargo, finalmente se obtuvo una biblioteca combinatoria dinámica multivalente y se analizó con éxito mediante técnicas de HPLC-MS. El DCL, está basado en el intercambio de sulfuro para formar puentes di-sulfuro entre las diferentes unidades de azúcar. Esto fue posible gracias a la solubilidad en agua de las subunidades carboxilato y al uso de enlaces cortos entre los puntos de conexión del tiol y de la estructura central, evitándose la formación de enlaces intramoleculares. Formación de una librería dinámica. Las partes se conectan a través de enlaces disulfuros. Sin embargo, incluso cuando se controlaron los problemas mencionados anteriormente, la formación fiable y estable de los miembros de la librería era difícil debido a la desintegración sustancial durante la etapa de análisis. Por lo tanto, la posterior comparación de las afinidades de los miembros de la DCL no era posible. No obstante, los enfoques presentados ofrecen oportunidades para nuevos experimentos, que con una cuidadosa elección de las condiciones pueden conducir al éxito. Desafortunadamente, el marco temporal de esta tesis no lo permitió estudiar en detalle; había que seguir otras pistas más prometedoras. La coordinación de ligando metal, especialmente con ligandos de tipo bipiridina coordinados a centros de FeII, evitó la mayoría de problemas encontrados anteriormente (parte desarrollada en el Capítulo 3). Observándose que en las condiciones necesarias para trabajar con la proteína elegida (ConA lectina) la formación de complejos era muy fiable. Como primera prueba de concepto para un comportamiento de dinámica combinatoria, se evaluaron DCLs simples que no contenían azúcares sobre la base coordinativa bipyridina. Una librería dinámica basada en el intercambio de ligandos de un centro metálico. Después, DCLs que contenían azúcares fueron sintetizadas y fueron comprobadas con la proteína. Ligandos con sustituyentes azúcar fueron usados como bloques prefabricados, obteniéndose, mediante síntesis sencillas y con buenos rendimientos. Ligandos con sustituyentes azúcares y basados en bipyridina que pueden formar complejos hexavalentes. Métodos de HPLC bien elegidos permitieron el análisis de los DCLs, así como la determinación de las afinidades relativas con la lectina ConA. La cuantificación de las entidades con más afinidad apoyó el concepto de multivalencia para sistemas que intercambian dinámicamente múltiples unidades de reconocimiento. A partir de este estudio básico, se desarrollaron otras DCLs que incorporaron componentes de diferentes geometrías. Las afinidades relativas de estos complejos compararon y revelaron que algunos miembros de la biblioteca contienen disposiciones tridimensionales más afines para la interacción con la lectina. La proteína tiene más afinidad a un único miembro de la biblioteca dinámica Por otra parte, los miembros de la librería de forma esférica parecen mostrar mayor afinidad a la proteína, en acuerdo con la teoría de “statistical rebinding”. Una biblioteca dinámica de geometrías diferentes. En resumen, DCLs basadas en la coordinación con metal (en contraste con enlaces covalentes dinámicos) han demostrado que constituyen una manera fácil de acceder a los procesos de intercambio multivalentes, proporcionando nuevas perspectivas para desentrañar las reglas de interacciones multivalentes de azúcares - lectina.

Le présent projet vise à analyser l'influence de la multivalence dans les interactions sucres-lectines. En collaboration avec des équipes externes, une étude par microscopie à force atomique (AFM) de l'interaction entre des ligands synthétiques de différentes valences et leurs lectines spécifiques a été entreprise. Dans le cadre de cette étude, une première caractérisation fondamentale de l'interaction sucre-lectine a été menée. Cette caractérisation concerne plus particulièrement l'influence de la multivalence sur les forces d'adhésion et la dynamique de l'interaction entre les ligands synthétiques multivalents et une lectine modèle, la lectine d'arachide PNA. Une seconde caractérisation, d'aspect plus appliqué, concerne l'utilisation des ligands synthétiques multivalents dans une approche thérapeutique antiadhésive pour le traitement des infections urinaires chroniques dues à Escherichia coli uropathogène (UPEC). Le caractère innovant des ligands (obtenus par une synthèse chimique rationnelle) ainsi que l'approche utilisée pour caractériser leurs interactions avec les lectines à l'échelle moléculaire par AFM témoigne de l'originalité du projet.

Virtually all biologically relevant functions and processes are mediated by non-covalent, molecular recognition events, demonstrating astonishingly diverse affinities and specificities. Despite extensive research, the origin of affinity and specificity in aqueous solution - specifically the relationship between ligand binding thermodynamics and structure - remains remarkably obscure and is further complicated in the context of multivalent interactions. Multivalency describes the combinatorial interaction of multiple discrete epitopes across multiple binding surfaces where the association is considered as the sum of contributions from each epitope and the consequences of multivalent ligand assembly. Gaining the insight necessary to predictably influence biological processes with novel therapeutics begins with an understanding of the molecular basis of solution-phase interactions, and the thermodynamic parameters that follow from those interactions. Here we continue our efforts to understand the basis of aqueous affinity and the nature of multivalent additivity.

Multivalent additivity is the foundation of fragment-based drug discovery, where small, low affinity ligands are covalently assembled into a single high affinity inhibitor. Such systems are ideally suited for investigating the thermodynamic consequences of multivalent ligand assembly. In the first part of this work, we report the design and synthesis of a fragment-based ligand series for the Grb2-SH2 protein and thermodynamic evaluation of the low affinity ligand fragments compared to the intact, high affinity inhibitor by single and double displacement isothermal titration calorimetry (ITC). Interestingly, our investigations reveal positively cooperative multivalent additivity - a binding free energy of the full ligand greater than the sum of its constituent fragments - that is largely enthalpic in origin. These results contradict the most common theory of multivalent affinity enhancement arising from a "savings" in translational and rotational entropy. The Grb2-SH2 system reported here is the third distinct molecular system in which we have observed enthalpically driven multivalent enhancement of affinity.

Previous research by our group into similar multivalent affinity enhancements in protein-carbohydrate systems - the so-called "cluster glycoside effect" - revealed that evaluation of multivalent interactions in the solution-phase is not straightforward due to the accessibility of two disparate binding motifs: intramolecular, chelate-type binding and intermolecular, aggregative binding. Although a number of powerful techniques for evaluation of solution-phase multivalent interactions have been reported, these bulk techniques are often unable to differentiate between binding modes, obscuring thermodynamic interpretation. In the second part of this work, we report a competitive equilibrium approach to Molecular Recognition Force Microscopy (MRFM) for evaluation of ligand binding at the single-molecule level with potential to preclude aggregative associations. We have optimized surface functionalization strategies and MRFM experimental protocols to evaluate the binding constant of surface- and tip-immobilized single stranded DNA epitopes. Surprisingly, the monovalent affinity of an immobilized species is in remarkable agreement with the solution-phase affinity, suggesting the competitive equilibrium MRFM approach presents a unique opportunity to investigate the nature of multivalent additivity at the single molecule level.

Dissertation

abstract: Multivalency is an important phenomenon that guides numerous biological interactions. It has been utilized in design of therapeutics and drug candidates. Hence, this study attempts to develop analytical tools to study multivalent interactions and design multivalent ligands for drug delivery and therapeutic applications. Atomic Force Microscopy (AFM) has been envisioned as a means of nanodiagnostics due to its single molecule sensitivity. However, the AFM based recognition imaging lacks a multiplex capacity to detect multiple analytes in a single test. Also there is no user friendly wet chemistry to functionalize AFM tips. Hence, an uncatalyzed Click Chemistry protocol was developed to functionalize AFM tips. For multiplexed recognition imaging, recognition heads based on a C3 symmetrical three arm linker with azide functionalities at its ends were synthesized and the chemistry to attach them to AFM tips was developed, and these recognition heads were used in detecting multiple proteins simultaneously using AFM. A bis-Angiopeptide-2 conjugate with this three-arm linker was synthesized and this was conjugated with anti-West Nile virus antibody E16 site specifically to target advanced West Nile virus infection in the Central Nervous System. The bis-Angiopeptide-2 conjugate of the antibody shows higher efficacy compared to a linear linker-Angiopeptide-2 conjugate of the antibody in in vitro studies and currently the efficacy of this antibody conjugate in studied in mice. Surface Plasmon Resonance imaging (SPRi) results indicate that the conjugation does not affect the antigen binding activity of the antibody very significantly. A Y-shaped bisbiotin ligand was also prepared as a small sized antibody mimic. Compared to a monovalent biotin ligand, the y-Bisbiotin can cooperatively form a significantly more stable complex with streptavidin through intramolecular bivalent interactions, which were demonstrated by gel electrophoresis, SPR and AFM. Continuing on these lines, a four-arm linker was synthesized containing three single chain variable fragments (scFv) linked to the scaffold to form a tripod base, which would allow them to concomitantly interact with a trimeric Glycoprotein (GP) spike that has a “chalice” configuration. Meanwhile, a human IgG1 Fc is to be installed on the top of the tetrahedron, exerting effector functions of a monoclonal antibody.
Dissertation/Thesis
Doctoral Dissertation Chemistry 2016

Self-healing macromolecular structures, submicron capsules and fibers with molecular recognition, stimuliresponsive molecules, solvent-free rheological reversibility, multivalency in rational drug design, and the emergence of new fields of adaptive and evolutive chemistry will require a predictive synergy of tailored non-covalent and covalent bonding in molecular design. Supramolecular chemistry has emerged as a stimulating focal point that will enable these scientific and technological discoveries, and biorecognition and biomolecular organization often serve as the inspiration for the future design of supramolecular assemblies. Linear and branched macromolecules are conventionally prepared using unique combinations of step-growth and chain polymerization strategies wherein the repeating units are irreversibly connected using stable covalent bonds. Moreover, optimum physical properties and commercial success of macromolecules are derived from our ability to prepare exceptionally high molecular weights in a controlled fashion. Although high molecular weight linear macromolecules are desirable for the optimization of physical performance and commercial impact, high molecular weights often compromise future solvent-free manufacturing, melt processability, thermal stability, and recyclability of the final products. Our recent efforts have demonstrated the utility of living anionic polymerization techniques to place functionality at desired positions on the polymer backbone. This control allowed investigation of the relationship between topology and tailored functionality, a fundamental investigation that may lead to interesting adaptive and smart applications. Specifically, the synthesis of polyisoprene homopolymers in a variety of topologies was performed, as well as the introduction of complementary hydrogen bonding to diverse families of hydroxyl containing polymeric and monomeric precursors.