Introduction

Chemical biologists seek to design new chemical tools for use in research and medicine. Their search is predicated on the incredible diversity of chemical structures, both natural and otherwise. This diversity was well represented at the Chemical Biology Discussion Group's Special Year-End Meeting, held June 2, 2008. Keynote speaker Michael Famulok of the Universität Bonn was joined by six graduate students from the New York area in a dazzling display of the diversity of chemical tools and targets under pursuit by those in the chemical biology arena. The meeting was organized by Paramjit Arora of New York University.

Aptamers were used to identify inhibitors of HIV replication.

Molecules represented ranged from nucleic acids to peptides to small drug-like compounds. Famulok described his work using aptamers, small, highly structured nucleic acids, to screen small molecule libraries for potential drug compounds. Famulok has used aptamers to identify new inhibitors of human immunodeficiency virus (HIV) replication, as well as inhibitors of a class of proteins involved in cytoskeletal remodeling known as cytohesins. His work on cytohesins has identified interesting new roles for these proteins in insulin receptor signaling and metabolic regulation.

Famulok was followed by Yu Liu of New York University, whose work involves the formation of DNA catenanes, interlocking ring structures that can be used to attach a label or other useful functional component to a DNA molecule. These interlocking ring structures, formed by incorporating 2′-modified nucleoside monomers into DNA sequences followed by DNA templated 2′,2′-coupling, may be useful for the detection and manipulation of DNA molecules. Catenanes have potential research applications, as well as potential uses in the control of gene expression for gene therapy.

The focus then turned to protein engineering, as Philip Effraim of Columbia University described his work on the recognition of aminoacylated tRNAs by the protein translation machinery. Effraim is gaining new insight into the kinetics of translation and elongation by studying the incorporation of amino acids that are coupled to the wrong tRNA. Besides shedding light on a fundamental biological process, this work may improve chemical biologists' ability to incorporate unnatural amino acids into engineered proteins to add new chemical functions.

Elizabeth George Cisar of the Rockefeller University presented her work on the mechanism of activation of AgrC, a histidine kinase receptor involved in quorum sensing in Staphylococcus aureus. The population density of this pathogen affects the expression of virulence factors via a set of genes and proteins known as the accessory gene regulator quorum sensing system. Cisar investigated whether AgrC behaves like other histidine kinase receptors, functioning as a dimer in which each subunit phosphorylates the other.

Justin Cisar of Memorial Sloan-Kettering Cancer Center discussed work that has led to the development of small molecule inhibitors that are highly specific for the enzymes that make nonribosomal peptides, important determinants of bacterial virulence. These peptides are synthesized from amino and aryl acid building blocks by enzymes known as nonribosomal peptide synthetases (NRPSs). Cisar and his colleagues have designed nonhydrolyzable intermediate analogs that bind tightly to the amino acid adenylation domain of these enzymes. Such intermediates have potential applications as antimicrobial agents.

Elizabeth Harker of Yale University presented her work on refining the structures of synthetic β-peptides designed to inhibit HDM2, a protein involved in certain cancers. HDM2 binds to the tumor suppressor p53, interfering with its role in regulating cell division and death. The synthetic β-peptides are designed to prevent HDM2 from binding to p53. Harker is engaged in refining the peptides' structures so that they can enter cells more easily and are also less toxic, improving their potential as therapeutic molecules.

Finally, Keith Baessler of Stony Brook University described his work on protein recognition events in mammalian fertilization, which he has carried out with the aid of oligomeric peptide polymers. He is investigating whether binding of fertilin β by β1 integrin is important for egg and sperm recognition events. These studies could allow the design of new agents that promote human fertility or, conversely, new contraceptives.

The ultimate targets of this research ranged from AIDS to bacterial infection to cancer to infertility, in an impressive display of the far-reaching powers of the new science of chemical biology. The session was particularly noteworthy in that many of these research projects represented next generation approaches, built on the initial successes of a wide variety of imaginative ideas. These young researchers also represent the next generation of chemical biologists who will carry these ideas to fruition, whether in the development of new therapeutics or in the investigation of previously difficult to study biological problems.

Student and Postdoc Presentations

Interlocking Molecules by Coupling Across a DNA Duplex Helical Turn

Speaker: Yu Liu
New York University

Yu Liu is a member of James Canary's laboratory group at New York University. Canary and his group are interested in the design, synthesis, and testing of new chemical compounds for use in nanotechnology as well as in biological research applications. Among other projects, they have designed DNA-polyamide ladder polymers. These polymers use DNA molecules as templates for the assembly of industrial polymers, such as nylon, with defined sizes, sequences, and topologies.

Liu described his work on the synthesis of polymer components that can be coupled across a full turn of the helix along a double-stranded DNA molecule. Specialized DNA phosphoramidites were synthesized to contain long side chains. After incorporation into the DNA, these side chains could be linked across a single turn of the DNA helix with a complementary DNA circle as a template. The net effect of this coupling was to form two interlocking rings, one a DNA circle and the other a hybrid DNA-organic macrocycle. This interlocking ring structure is known as a catenane.

Formation of a catenane creates in effect a molecular "padlock" on the DNA molecule, which can be used to attach a label or other useful functional component. Liu has designed a photocleavable linkage that could be used to remove the second ring if desired, as well as a ring containing molecular "tails" that can be used to chemically change its functionality after synthesis of the catenane. These interlocking ring structures have many potential uses in biological research for the detection and manipulation of DNA molecules, and might also play a role as part of a gene therapy strategy for medical purposes.

Aminoacyl-tRNA Substrate Specificity of the Translation Machinery

Speaker: Philip Effraim
Columbia University

Philip Effraim is an MD-PhD student in Virginia Cornish's laboratory at Columbia University. Cornish and her group are interested in protein engineering as a means to understanding protein function at the molecular level. One useful method for exploring the relationship between structure and function is to make proteins that include amino acids with functional groups not normally found in nature, also known as unnatural amino acids.

Unnatural amino acids can be incorporated into proteins by substituting them onto tRNAs in the place of natural amino acids (misacylation) and allowing them to be incorporated via the ribosome in the normal course of protein translation. However, unnatural amino acids attached to these misacylated tRNAs often are not incorporated into proteins with the same efficiency as natural amino acids. This observation calls into question one of the fundamental tenets of translation, the adapter hypothesis, which holds that the anticodon portion of the tRNA controls specificity of amino acid incorporation without regard to the amino acid attached. It suggests that the ribosome recognizes the whole structure of an acylated tRNA rather than just its anticodon.

Effraim is investigating this question in detail by studying the effects of misacylated tRNAs on the kinetics of protein translation and elongation, a process that normally proceeds very rapidly and with a very low error rate. He has begun by studying the incorporation of natural amino acids that are attached to the wrong tRNA, for example, a phenylalanine tRNA carrying an alanine instead, or vice versa, in competition with the correctly acylated tRNAs. However, using two types of assays, he has found no significant differences in the ability of the protein translation machinery to handle misacylated tRNAs when they are carrying natural amino acids. This finding suggests that for natural amino acids, the translation machinery has evolved to accept any amino acid rather than allow the translation process to come to a halt with potentially deleterious effects on cell function.

Effraim is planning to investigate the effects of other misacylated tRNAs with more drastic structural variations, for example, containing proline, which is an imino acid rather than an amino acid. These experiments are likely to shed important light on the mechanisms of protein translation, and may also lead to more efficient ways of producing proteins that contain unnatural amino acids with new and interesting functionalities.

Mechanism of Signal Transduction by a Staphylococcal Quorum Sensing Receptor

Speaker: Elizabeth George Cisar
The Rockefeller University

Elizabeth Cisar is a graduate fellow at the Rockefeller University, working in the laboratory of Tom Muir. One project in the Muir lab is the study of quorum sensing in Staphylococcus aureus, an opportunistic pathogen much in the news of late as a threat to human health. This project has already begun to shed light on an interesting problem in biodevelopment, and may also lead to potential medical applications in the design of new antimicrobials.

Cisar is investigating the accessory gene regulator (agr) quorum sensing system, which coordinates the production of virulence factors with changes in S. aureus population density. This system relies on secreted peptides that either activate or inhibit virulence, depending on which strain of S. aureus they encounter. The peptides affect virulence by binding to a receptor histidine kinase known as AgrC. Peptide binding causes AgrC to autophosphorylate, leading to the phosphorylation of an intracellular response regulator, AgrA, and subsequent activation of transcription of the agr gene cluster, including the important regulatory RNA, RNAIII.

Inhibition of Nonribosomal Peptide Synthetase Amino Acid Adenylation Domains

Speaker: Justin Cisar
Memorial Sloan-Kettering Cancer Center

Justin Cisar is a member of Derek Tan's laboratory at Memorial Sloan-Kettering Cancer Center. Tan's group studies nonribosomal peptides, natural products synthesized by bacteria and fungi that are involved in virulence, iron acquisition, commensalism, and biofilm generation. They are designing small molecule probes that could be used to study the synthesis of these peptides or as starting points for future antimicrobials.

As the name implies, nonribosomal peptides are not synthesized by the ribosome but instead are synthesized from amino and aryl acid building blocks by enzymes known as nonribosomal peptide synthetases (NRPSs). These enzymes carry out a multistep process involving adenylation of the amino acid, acyl transfer to a carrier domain, modification and extension of the peptide backbone and finally product release. Tan and his group are focusing on the first steps of this process, which are carried out by the adenylation domain of the NRPSs. These steps depend on the formation of a tightly bound aminoacyl-AMP intermediate. One strategy for disrupting this process is to design a nonhydrolysable intermediate that binds tightly but cannot be cleaved, preventing further progression through the catalytic cycle.

Tan and his group had already successfully designed one such inhibitor, known as salicyl-AMS, which inhibits the synthesis of salicyl-capped siderophores. Siderophores are nonribosomal peptides that act as iron scavengers. They are important for virulence in such pathogens as Yersina pestis and Mycobacterium tuberculosis, the causative agents of plague and tuberculosis, respectively. This inhibitor, while successful, has limited applications because it only affects the production of nonribosomal peptides that are salicylated.

In order to make a more universal inhibitor, Cisar has synthesized an inhibitor that targets amino acid adenylation, a step required for the synthesis of all nonribosomal peptides. Such inhibitors are already available but are of limited utility because they also inhibit the aminoacyl tRNA synthetases required for protein translation by the host organism. Cisar synthesized a new inhibitor, cyclo-L-alanyl-AMS, based on the single known structure of an amino acid adenylation domain, a phenylalanine adenylation domain crystallized with phenylalanine and AMP bound. This structure shows an reaction intermediate that adopts a bent, cisoid conformation, in contrast to the extended, transoid conformation adopted by reaction intermediates found bound to aminoacyl tRNA synthetases. Cisar has shown that the cisoid configuration of this new inhibitor specifically inhibits NRPSs while sparing host aminoacyl tRNA synthesis. This higher specificity should allow the design of new antimicrobials based on this structure.

β-peptide Ligands for hDM2: Cellular Entry and p53 Activation

Speaker: Elizabeth Harker
Yale University

Elizabeth Harker is a member of Alanna Schepartz' laboratory group at Yale University. Schepartz' research concerns the design of small stable peptides and other molecules for use as molecular probes or therapeutics. Harker described her work with a group of peptides synthesized from β-3-substituted amino acids, known as β-peptides. These peptides form helices that are very similar in geometry to α-helices but are very stable because they are not hydrolyzed by endogenous proteases.

Design of β-peptides targeting the p53-HDM2 interaction.

One protein–protein interaction that has been targeted by the Schepartz group is the p53-HDM2 (human homologue of murine mdm2) interaction. HDM2 is the principal cellular antagonist of the tumor suppressor protein p53. HDM2 is overexpressed in some cancers, leading to suppression of p53, preventing regulation of apoptosis and the cell cycle and thus disrupting the control of cell growth and division. The interface between p53 and HDM2 has been crystallized, and Schepartz' group has designed a series of β-peptides that mimic this interaction, binding to HDM2 in the place of p53 and preventing it from suppressing p53 activity.

Harker has improved the binding of these initial peptides to HDM2 by synthesizing new forms that incorporate unnatural amino acid side chains. She is also working on modifying these peptides to improve their ability to penetrate into cells, since the original peptides required a special reagent to allow them to enter the cytoplasm. The new series of peptides retains their high affinity for the HDM2 target, persist well in the cell due to high proteolytic resistance, and enter the cell much more easily than first generation peptides. However, these new peptides are also more toxic to cells, necessitating further studies to optimize the structure and function of these peptides before they can be used as anticancer therapeutics.

Probing the Mechanisms Behind Inhibition of Fertilization and Activation of Development by Fertilin-β derived Oligopeptide Polymers

Speaker: Keith Baessler
Stony Brook University

Keith Baessler is investigating the mechanisms by which mammalian sperm bind to and fuse with the egg plasma membrane during the process of fertilization, as a member of Nicole Sampson's laboratory at Stony Brook University. This work has potential implications for both fertility treatments and in the design of future methods of population control.

Baessler is studying a protein carried by sperm known as fertilin β, which appears to be important for recognition events occurring between the sperm and the egg during fertilization. Fertilin β is a member of a group of proteins known as ADAM proteins, for "a disintegrin and metalloprotease domain" protein. ADAM proteins are well conserved among mammals, carrying a conserved tripeptide ECD motif. This motif is found in the disintegrin domain, and believed to confer on the protein the ability to bind to membrane-bound receptors on the egg known as integrins.

Multiple lines of evidence suggest that the β1 integrin found on egg cells acts as a receptor for sperm fertilin β and that this interaction is important for fertilization. However, this interpretation has been called into question by a number of findings, including the finding that knockout mice lacking β1 integrin are still fertile. Baessler has been investigating this question with the use of oligomeric peptide polymers containing the ECD motif. He presented studies showing that polymers carrying multiple copies of ECD strongly inhibit fertilization in wild-type eggs but not in eggs from β1 integrin knockout mice. Baessler's studies suggest that the fertilinβ/β1 integrin interaction is important for sperm recognition under normal circumstances but that the knockout mice have developed a compensatory mechanism that still allows fertilization. Further study will be needed to solidify our understanding of this important event in the fertilization process.