BiOther Other Contributorapeutics: Recent Developments using Chemical and Molecular Biology: Volume 36 (Drug Discovery Series) - Hardcover

Hardcover

ISBN 10: 1849736014 ISBN 13: 9781849736015

Publisher: Royal Society of Chemistry, 2013

Synopsis

BiOther Other Contributorapeutics are often considered to be beyond the reach of the medicinal chemist, but this book demonstrates that chemistry has an essential role in the future success of this area.

About this title

From the Back Cover

This book will illustrate the successful partnership of chemistry and biology to advance successful biotherapeutic modalities. Molecular design to create function is common to both chemical and molecular biology, and this text will highlight recent developments from these disciplines that have delivered drugs, clinical candidates or significantly advanced biotherapeutic approaches.

Biotherapeutics are often considered to be beyond the reach of the medicinal chemist, but this book demonstrates that chemistry has an essential role in the future success of this area, by explaining and describing the chemical biology technologies that underpin specific therapeutic advances and demonstrating the unique value of molecular design and understanding.

Covering topics such as selective protein modification, immunopharmacotherapy, chemically programmed vaccinations, nanobodies and antibodies, this book will provide essential reading for medicinal and pharmaceutical chemists working in both industry and academia.

Excerpt. © Reprinted by permission. All rights reserved.

Biotherapeutics

Recent Developments using Chemical and Molecular Biology

By Lyn H. Jones, Andrew J. McKnight

The Royal Society of Chemistry

Copyright © 2013 The Royal Society of Chemistry
All rights reserved.

ISBN: 978-1-84973-601-5

Contents

Chapter 1 Synthetic Immunology Thihan R. Padukkavidana, Patrick J. MCEnaney and David A. Spiegel, 1, 
Chapter 2 Immunopharmacotherapy for Nicotine Addiction Jonathan W. Lockner and Kim D. Janda, 36, 
Chapter 3 Carbohydrate Vaccines Benjamin Schumann, Chakkumkal Anish, Claney L. Pereira and Peter H. Seeberger, 68, 
Chapter 4 Generation and Maturation of Therapeutic Antibodies via In Vitro Somatic Hypermutation David J. King, 105, 
Chapter 5 Synthetic Protein Biologics Benjamin G. Davis, 130, 
Chapter 6 Recent Advances in Antibody–Drug Conjugates Edmund I. Graziani and L. Nathan Tumey, 145, 
Chapter 7 MicroRNA Biotherapeutics: Key Challenges from a Drug Development Perspective Sterghios Athanasios Moschos, 176, 
Chapter 8 Novel Therapeutic Agents from Bacterial Toxins John A. Chaddock, 224, 
Chapter 9 Phage Selection of Mono- and Bicyclic Peptide Ligands Shiyu Chen and Christian Heinis, 241, 
Chapter 10 Chemical Approaches for Localization, Characterization and Mimicry of Peptide Epitopes Paul R. Werkhoven and Rob M.J. Liskamp, 263, 
Chapter 11 Cystine-Knot Micro-Proteins Bill Eldridge, Simon Robins and Duncan McGregor, 285, 
Subject Index, 298, 

CHAPTER 1

Synthetic Immunology

THIHAN R. PADUKKAVIDANA, PATRICK J. MCENANEY AND DAVID A. SPIEGEL

1.1 Introduction and Scope

The field of immunology has become increasingly lucid at the level of atoms and molecules. Owing also to advances in synthetic chemistry, the rational design and construction of synthetic systems that perform complex immunological functions – an area termed synthetic immunology – has come within reach. Here we will highlight one facet of synthetic immunology concerned with the development of low-molecular weight ("small"), synthetic molecules that are capable of functionally mimicking biological molecules. It is important to note at the outset that this article does not aim to be comprehensive in scope. At the expense of being all-inclusive, we focus on several specific contributions, which highlight how advances in immunology and chemistry have proven mutually complementary. We have divided this chapter into four subsections: (1) synthetic ligands for pattern recognition receptors, including toll-like receptors (TLRs), NOD-like receptors (NLRs), nuclear family receptors and C-type lectins, (2) synthetic molecules that modulate the complement system, (3) synthetic systems for controlling cell–cell communication, including chemokine/cytokine mimetics, and (4) synthetic ligands for modulating adaptive immune processes, including T-cell and B-cell functions.

We regret not being able to cover all of the exciting developments that might be classified into the area of Synthetic Immunology. Such areas include: synthetic vaccine development, as this topic is covered elsewhere in this book; synthetic modulators of cellular signaling processes, as the functions of such molecules extend beyond the immune system; protein-based and cellular immunotherapies, including therapeutic monoclonal antibodies; immunomodulator strategies involving nanoparticles or virus-like particles; DNA- and RNA-based therapeutics; and strategies for controlling cellular differentiation. Lead references to each of these areas are provided for interested readers.

It is our hope that this chapter will serve as a broad-based introduction for biomedical scientists, including chemists interested in extending their activities into the immunological realm, as well as immunologists looking to learn about how modern synthetic chemistry can enhance fundamental biological understanding. Ultimately, we believe that the intellectual perspective residing at the interface between synthetic chemistry and immunology will enable scientific advances that were never before thought possible, thus proving critical to the furtherance of basic biomedical research and patient care.

1.2 Synthetic Ligands for Pattern Recognition Receptors (PRRs)

Pattern recognition receptors (PRRs) are a diverse class of proteins that function canonically as part of the innate immune response. These receptors recognize pathogen-associated molecular patterns (PAMPs), which are widely conserved, repeating motifs found within pathogens and not within hosts, as well as damage-associated molecular patterns (DAMPs), which are host-derived molecules that arise from tissue damage. Improper activation of these receptors has been shown to cause hyper-inflammatory disease states, and extensive research efforts have focused on identifying PRR antagonists. Such developments have been reviewed elsewhere. In this section we discuss a few select examples of molecules that functionally mimic the natural ligands of PRRs.

1.2.1 Synthetic Mimics of TLR Ligands

Toll-like receptors (TLRs) were originally discovered as important receptors for Drosophila melanogaster embryonic development, and were later found to play a critical role in innate immunity in humans. Extensive research during the past two decades has revealed the TLR superfamily to contain more than ten different family members (TLRs 1–13).

TLR2 is capable of binding various bacterial lipopeptides, as well as self-derived ligands such as high mobility group box 1 (HMGB1) and products of lipid oxidation. Interestingly, TLR2 preferentially forms heterodimers with other TLR members (e.g., TLRs 1 and 6), thus expanding its ligand binding profile. Indeed, synthetic lipopeptide collections, designed to mimic the acylated amino terminus of bacterial lipoproteins, have proven critical to understanding the different adaptor molecules involved in TLR2 function. These studies have led to the understanding that the TLR2 heterodimer formed in response to a given ligand depends both on the arrangement of fatty acyl groups and the sequence of peptide backbones in that ligand. For example, synthetic tri-acylated lipopetpides, such as Pam3CSK4 (Figure 1.1, Panel A, 1), selectively engage the TLR2–TLR1 heterodimer, while di-acylated analogs, such as Pam2CSK4 (2), bind both TLR2–TLR1 and TLR2–TLR6 heterodimers. Modification of the peptide backbone in Pam2CSK4 with the decapeptide GDPKHSPKSF provides fibroblast-stimulating lipopeptide-1-(FSL-1, 3), which is selective for the TLR2–TLR6 complex. A crystal structure of the TLR1–TLR2–lipopeptide provides some insight into the molecular origin of this ligand-dependence. Furthermore, the specific TLR2 heterodimer formed in response to ligand stimulation can have a significant effect on downstream biological outputs. For example, synthetic ligands that induce TLR2–TLR1 heterodimer formation (e.g., Pam3CSK4) have been shown to enhance regulatory T-cell (Treg) proliferation to a larger extent than those leading to other heterodimeric TLR2 complexes. Conversely, a small molecule inhibitor of the TLR2–TLR1 complex was recently discovered through a cell-based high-throughput small molecule screen. Efforts to expand the repertoire of TLR2-targeted compounds offer hope of providing chemical insights into immunomodulatory functions, while also delivering useful lead molecules for studying pathogen recognition, cancer progression, and other disease-relevant biological processes.

TLR4 recognizes lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell wall. Stimulation of TLR4 leads to rapid induction of various pro-inflammatory processes including cytokine release and immune cell differentiation. TLR4 ligands can serve as useful adjuvants for vaccine development, however activation of this receptor is also associated with autoimmune diseases, neuropathies, and septic shock. Therefore there has been significant interest in identifying both synthetic agonists and antagonists of TLR4. Efforts to this end have primarily started from lipid A (Figure 1.1, Panel B, 4) – the hydrophobic portion of LPS – and have led to the development of numerous synthetic immunomodulators. For example, early studies demonstrated that removal of the anomeric phosphate group in 4 can ameliorate some of the molecule's toxic effects in humans. The resulting compound, monophosphoryl lipid A (MPL, 5), retains its TLR4 agonist activity and has been approved by the FDA, for use as a vaccine adjuvant. Further investigations into synthetic lipid A analogs have led to a general model relating ligand structure with TLR4-modulating activity: agonists are believed to form "conical" structures in solution – wherein the cross-section of the hydrophobic portion of the molecule is greater than that of the hydrophilic one – whereas antagonists are believed to form "cylindrical" shapes such that hydrophobic and hydrophilic cross-sections are nearly identical. Indeed, this model is supported by extensive structure-activity relationships along with recent crystallographic studies. For example, compounds lacking hydrophobic acyl substituents – such as eritoran (Figure 1.1, Panel C, 6) – serve as receptor antagonists. On the other hand, agonist structures have been obtained from replacement of labile phosphate moieties with negatively charged bioisosteres (e.g., sulfate), substitution of ester-branched acyl groups with corresponding alkyl ethers, and removal of the lipid A reducing sugar to afford aminoalkyl glucosaminide 4-phosphates (AGPs, 7). Complete replacement of the lipid A disaccharide with a simple phosphatidylethanolamine-based construct provides agonist E6020 (8), which is believed to retain the "conical" conformation and is capable of eliciting TNF-α production from whole blood, as well as IL-6 production and NF-κB activation in cell lines expressing TLR4. Additional TLR4 modulators have been obtained using "glycocluster" strategies, attaching LPS to magnetic nanoparticles, and other approaches. Furthermore, the discovery of TLR4-active compounds structurally unrelated to lipid A, such as paclitaxel, heme derivatives, and opioids, will undoubtedly lead to new opportunities for developing synthetic immunomodulators.

TLR7/8 are expressed in a variety of immune cells of the myeloid lineage and are intimately involved in anti-viral immunity by triggering the release of type 1 interferons. The first synthetic ligands for TLR7/8 were discovered by 3M scientists in cellular screens of nucleoside analogs for antiviral applications. Interestingly, although these efforts led to the development of the class of imidazoquinoline derivatives – including Imiquimod (Figure 1.2, Panel A, 9), which was first marketed in 1997 for treating complications of human papilloma virus (HPV) infection – it was not until much later that the mechanism of these compounds as TLR7/8 agonists was confirmed. Over the past decade, derivatives of imidazoquinolines, including resiquimod (10) and S-27609 (11), have proven to be effective TLR7-stimulating adjuvants resulting in anti-tumor activity and polarization of immune cells towards TH1 responses. Like single-stranded RNA (ssRNA), which is the natural ligand for TLR7/8, small molecule agonists such as 9–11 activate macrophages to produce pro-inflammatory cytokines (IFN, TNF-α and others) via the TLR7 MyD88-dependent signaling pathway. Furthermore, homology modeling studies suggest that both ssRNA and small molecule 10 have overlapping binding sites on TLR8, suggesting that imidazoquinolines are both structural and functional mimics of natural ligands. Further progress in illuminating the structural details of TLR7/8 interactions with their ligands will undoubtedly lead to improvements in synthetic ligand mimics.

Like TLR7/8, TLR9 is localized to endolysosomal compartments, recognizes foreign nucleic acids, and induces a type-1 IFN response upon activation. Unlike TLR7/8, however, the natural ligand for TLR9 is unmethylated CpG oligodeoxynucleotides (CpG DNA), which is present in both bacterial genomes and DNA viruses. Synthetic agonists for TLR9 that take advantage of structural similarities with natural nucleic acid ligands have been developed. For example, having determined that an accessible 5'-end of CpG-containing oligodeoxynucleotides is necessary for immunomodulatory activity of synthetic TLR9 ligands, researchers at Hybridon have developed a class of "immunomers" (12) containing short CpG DNA sequences chemically connected through synthetic 3'–3' linkers. Interestingly, both the chemical composition and the length of "immunomer" derivatives were found to impact their immunomodulatory effects. Indeed, immunomers containing only 5 or 6 nt in each segment possess immunostimulatory properties in both mice and humans. Also, replacement of phosphate backbone with phosphorothioate motifs, or incorporation of 5'-dinucleotides containing non-natural bases, can confer enhanced stability and alter patterns of cytokine secretion in tissue culture systems. Evaluation of synthetic TLR9 agonists in human clinical trials as vaccine adjuvants, and immunotherapies for allergy, cancer, and infectious disease are currently underway.

1.2.2 Synthetic NOD Agonists

Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are a family of soluble cytosolic proteins consisting of over twenty members. NLRs are believed to detect cytosolic PAMPs derived from foreign agents as well as danger-associated molecular patterns (DAMPs) from injured host tissue. NLRs promote NF-κB and mitogen-activated protein kinase (MAPK) signaling and complex with the inflammasomes to upregulate proinflammatory IL-18, IL-1β, IL-6, TNF, and type I IFN secretion.

NOD1 has been shown to recognize the dipeptide, D-glutamyl-mesodiaminopimelic acid (iE-DAP), present as a component of bacterial peptidoglycan (13), which activates the NF-κB pathway, enhancing the production and secretion of several cytokines. Hasegawa and coworkers have chemically synthesized iE-DAP derivatives to identify minimal motifs responsible for NOD1 activation. It was found that the iE-DAP structure serves as a core motif for NOD-1 activation; functionalization of this group with substituted alkyl chains (14), benzamides (15, 16), or short peptides (17) enhances NOD-1 activation. The authors speculate that this enhancement may result from increased hydrophobicity of the molecules compared to the parent compound (18), which enhances their interactions with the cell membrane. Notably, compound 14 was found to be several hundred fold more active than the original iE-DAP. Moreover, it was found that iE-meso-DAP stereoisomer showed a 10 to 10,000 fold greater ability to stimulate NOD-1-mediated NF-κB activation as compared to other stereoisomers.

NOD2, a close relative of NOD1, is expressed in several phagocytic cells, including monocytes, granulocytes, and dendritic cells. Its natural ligand is believed to be the MurNAc-L-Ala-D-isoGln muramyl dipeptide (MDP, Figure 1.3, Panel A, red box), which is found in both Gram-positive and Gram-negative bacteria. In a similar manner to NOD1, NOD2 ligation leads to the secretion of IL-12, IL-8, IL-6, and TNF-α. Almost four decades of research on MDPs have led to the development of numerous analogs, including mifamurtide (Figure 1.3, Panel C, 19), approved for combination chemotherapy treatments. One of the primary reasons for the development of these synthetic molecules was the potent pyrogenicity and somnogenicity induced by the natural ligand that made it relatively unattractive as an adjuvant. These derivatives of MDP have been shown to have greater biological activity and lack the undesirable characteristics of MDP, and have been extensively discussed elsewhere. Other efforts to optimize the biological effects of MDP have led to the identification of MDP-C (20), which is both apyrogenic and nonallergenic, yet potently immunostimulatory. Here, Yang et al. utilized a novel mesh-bagged gathered-bunch combinatorial chemistry strategy (abbreviated MBGB) to synthesize MDP analogs on solid supports. They produced over 2300 MDP derivatives and tested their ability to stimulate murine macrophages to eliminate tumor cells. This work has resulted in the discovery of the novel compound MDP-C (20), which significantly enhances dendritic cell (DC)-mediated, IL-2, IL-12 release and the induction of cytotoxic activity by cytotoxic T-cells (CTLs) in vitro. Finally, MDP-C has also been shown to have Hepatitis B Surface Antigen specific antibody response in vivo, with minimal pyrogenicity (in rabbits), passive cutaneous anaphylaxis (in rats), and low toxicity (in mice).

Researchers have also explored the use of cocktails of synthetic PRR agonists to stimulate NLRs and TLRs simultaneously. Such a combinatorial treatment may more closely mimic a bacterial infection compared to single agents, and in turn induce a potent immune reaction. Indeed, Tada et al. combined two synthetic NOD2 agonists (MDP and N-acetylmuramyl-L-aranyl-L-isoglutamine (MDP-LL)), two NOD1 agonists (FK565 and FK156), and various TLR agonists (TLR4- lipid A, TLR2- Pam3CSSNA, TLR3- poly (I:C), and TLR9- CpG DNA) to stimulate DC cultures. Combination treatments led to the synergistic upregulation of IL-12 and IFN-γ production by T-cells, indicative of a TH1-response. Utilizing complex cocktails to mimic the natural repertoire of PRR ligands is likely to prove increasingly important to synthetic immunology strategies in the future.

(Continues...)

Excerpted from Biotherapeutics by Lyn H. Jones, Andrew J. McKnight. Copyright © 2013 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2013
• Language: English
• ISBN 10: 1849736014
• ISBN 13: 9781849736015
• Binding: Hardcover
• Number of pages: 336
• Editor: Lyn H Jones