|I.||Expanding the Genetic Code|
|II.||Cell-Based Screens to Identify Small Molecules with Novel Biological Activities|
|III.||Exploring Molecular Diversity|
Expanding the Genetic Code
The genetic codes of every known organism specify the same 20 amino acid building blocks using triplet codons generated from A, G, C and T. These twenty amino acids contain a limited number of functional groups including acids, amides, alcohols, basic amines and thiols. Is this the ideal number or would additional amino acids allow the generation of proteins or even entire organisms with enhanced properties? The ability to site-specifically introduce amino acids with precisely tailored steric and electronic properties into proteins would also allow us to carry out "physical organic" studies of protein structure and function, much the same way as has been historically done with small molecules.
To this end, we have developed a methodology that allows one to genetically encode novel amino acids, beyond the common twenty, in both prokaryotic and eukaryotic organisms. This methodology involves the generation a unique codon-tRNA pair and corresponding aminoacyl-tRNA synthetase. Specifically, an orthogonal tRNA is constructed that is not a substrate for any natural aminoacyl synthetases and which inserts its cognate amino acid into the growing polypeptide chain in response to a nonsense or frameshift codon. A cognate synthetase is then generated which recognizes this unique tRNA and no other; the substrate specificity of this synthetase is then evolved to recognize a desired "twenty first" amino acid, and no endogenous amino acid. We have shown that this methodology can be used to efficiently incorporate a large number of unnatural amino acids into proteins in E. coli, yeast and mammalian cells with fidelity and efficiency rivaling that of the common 20 amino acids. These include heavy atom containing amino acids to facilitate x-ray crystallographic studies; amino acids with novel steric/packing and electronic properties; photocrosslinking amino acids which can be used to probe protein-protein interactions in vitro or in vivo; keto, acetylene, azide, and boronate containing amino acids which can be used to selectively introduce a large number of biophysical probes, tags, and novel chemical functional groups into proteins in vitro or in vivo; redox active amino acids to probe and modulate electron transfer; photocaged and photoisomerizable amino acids to photoregulate biological processes; metal binding amino acids for catalysis and metal ion sensing; amino acids that contain fluorescent or IR active side chains to probe protein structure and dynamics; α-hydroxy acids and D-amino acids as probes of backbone conformation and hydrogen bonding interactions; and sulfated amino acids and mimetics of phosphorylated amino acids as probes of posttranslational modifications.
We have also shown that one can "synthesize" a completely autonomous bacterium that not only genetically encodes a novel amino acid, but also biosynthesizes this amino acid from basic carbon sources. This is the first example of the creation of a twenty-one amino acid organism, and allows us to explore its ability to evolve under a variety of growth conditions. We are currently developing strategies to apply this methodology to multicellular organisms. A consensus-based approach has also been developed for generating new orthogonal tRNA-synthtase pairs that genetically encode novel amino acids in response to four base codons. In addition, in collaboration with the Church lab we are removing the redundancy in the existing genetic code of E. coli to encode additional amino acids. Ultimately, our hope is to ribosomally synthesize biopolymers composed only of unnatural amino acids. Finally, we are applying this methodology to studies of protein structure and function in vitro and in vivo, as well as the generation of proteins with novel properties, including therapeutic peptides, proteins and vaccines. For example, we have used unnatural amino acids: (1) as a mechanism to break immunological tolerance to self-proteins to generate vaccines and explore the underling mechanism of autoimmune disease; (2) to generate novel enzymes with "abiological cofactors"; (3) to generate antibodies and peptides with high affinity/selectivity for proteases and other extracellular proteins using phage-displayed libraries containing unnatural amino acids; (4) to generate bifunctional antibodies and antibody-drug conjugates as novel therapeutics (modified with ligands and other proteins); and (5) to generate novel biological probes (photocrosslinking agents, spectroscopic probes, photocaged polypeptides, etc.) to explore protein structure and function in vitro and in vivo. In collaboration with the Romesberg lab we have also added new building blocks to the DNA lexicon, and most recently are attempting to generate a strain of E. coli with DNA containing an unnatural base.
Cell-Based Screens to Identify Small Molecules with Novel Biological Activities
We have recently begun to use "rationally designed" chemical libraries together with phenotypic and pathway-based screens to identify and characterize small molecules with novel biological activities. Chemistries have been developed to efficiently synthesize large combinatorial libraries of heterocyclic compounds designed around a large number of molecular scaffolds, including substituted purines, pyrimidines, quinazolines, pyrazines, pyrrolopyrimidine, pyrazolopyrimidine, phthalazines, pyridazines, and quinoxalines. These libraries are being screened in a large number of cell-based assays to identify molecules that both modulate and provide new insights into complex cellular processes, and which ultimately may lead to new therapeutic agents for the treatment of unmet medical needs. For example image-based screens are being developed and carried out to identify molecules that control stem cell fate and self-renewal (embryonic and adult), as well as molecules that induce reprogramming of lineage committed cells. Molecules have been identified that: (1) selectively induce neurogenesis and oligodendrocyte formation in neural stem cells; (2) efficiently induce cardiomyogenesis or endodermal differentiation in ESCs; (3) selectively induce osteogenesis and chondrogenesis in mesenchymal stem cells; (4) allow embryonic stem cells to be propagated under chemically defined conditions; (5) induce the self-renewal of hematopoietic stem cells or their selective differentiation to erythrocytes or megakaryocytes; and (6) block the transdifferentiation of hepatic stellate cells to myofibroblasts. These molecules are currently being investigated for bone marrow transplants, and the treatment of osteoarthritis, multiple sclerosis, fibrosis and genetic blood diseases. They are also providing new insights into the complex biology of stem cells. For example, by using a combination of biochemical and genomic techniques (cDNA complementation, siRNA knockdown, photoaffinity labeling, phosphoproteome and mRNA expression analysis, etc.), we have shown that these molecules can block or activate key developmental pathways, selectively modulate nuclear localization of transcriptional activators, or antagonize nuclear receptors. We have also recently identified molecules that allow the reversible expansion of terminally differentiated cells (β cells, RPEs, cardiomyocytes) or reprogram lineage committed cells to other cell types.We are also carrying out cellular screens to identify molecules that may lead to the development of new therapeutics for cancer, metabolic and cardiovascular disease, infectious disease, and a number of orphan diseases. These include screens for molecules that modulate SMN2 splicing (SMA); β cell stress (Type 1 diabetes); epithelial-mesenchymal transition (cancer); fetal hemoglobin expression (sickle cell); cancer stem cell self-renewal, differentiation and apoptosis (cancer); Myc expression (cancer); M1/M2 macrophage differentiation, TNF secretion, and naive T-cell differentiation (inflammation and autoimmune disease); ENAC activity (cystic fibrosis) and Hippo Yap signaling (cardiac regeneration). In addition we have ongoing programs aimed at a number of neglected disease including tuberculosis, malaria, helminth infections and diarrheal diseases. Many of these programs are being carried out in collaboration with scientists at Calibr which has state of the art HT screening facilities, compound libraries, medicinal and protein chemistry and pharmacology.
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Exploring Molecular Diversity
One example of the synergy between chemistry and biology in the synthesis of new molecular function is the development and application of combinatorial strategies. This approach, in which large, diverse collections or "libraries" of molecules are generated and subsequently screened/selected for novel functions, stems from combinatorial processes in nature. For example, the humoral immune system has developed highly sophisticated combinatorial mechanisms for generating large libraries of antibodies and selecting those that can recognize foreign antigens with high affinity and selectivity. The notion that natural immunological diversity can be used to generate novel chemical function was illustrated by the generation of catalytic antibodies. The early experiments involved the generation of esterolytic antibodies using phosphonate/phosphate transition state analogues. Since those experiments, antibodies have been developed that catalyze a wide array of chemical and biological reactions, from acyl transfer to Diels-Alder reactions, with specificities rivaling or exceeding those of enzymes. In a number of cases, antibodies have been found to have rates and mechanisms comparable to those of known enzymes (e.g., catalysts for pericyclic, acyl transfer, metallation, and adol reactions). In addition to the notion of transition state stabilization, many other strategies have been developed to generate catalytic antibodies including general base and covalent catalysis, proximity effects, and substrate strain. Most recently, we have focused on the development of novel chemical screens, and genetic and phage-based selections for identifying mutants with enhanced catalytic function.The biochemical and structural characterization of catalytic antibodies is also providing insights into the mechanisms and evolution of binding and catalytic function in nature. For example, studies of a ferrochelatase antibody provided the first direct structural evidence for the Haldane substrate strain theory of catalysis. Structural comparisons of germline and affinity-matured antibodies provided new insights into the molecular nature of the immune response, including the critical role of structural plasticity in determining the tremendous binding potential of the germline repertoire. These structures have also revealed important insights in the role of somatic mutations removed from the active site in controlling active site structure, catalytic activity and the thermodynamic stability of antibodies. Most recently, we have begun to explore the engineering of antibodies with novel hypervariable loops to create a new class of growth factors and cytokines with enhanced pharmacological properties. The demonstration that the vast structural diversity of antibody molecules can be redirected with the proper chemical instruction to generate selective catalysts shows the synthetic power of using molecular diversity (the antibody repertoire in this case) to produce new function. We are applying this combinatorial approach to many other problems in chemistry and biology including the generation of novel growth factors, cytokines and signaling proteins with enhanced pharmacological properties; selective protein receptors for both small and macro-molecules; novel probes of cellular signaling; and artificial viroid like RNAs capable of rolling circle replication. We have also extended diversity-based approaches to the generation of solid state materials with novel properties. This has involved the development of methods for the parallel synthesis, processing and screening of large libraries of solid state inorganic and organic materials (electronic, magnetic, optical, and catalytic) and even devices for new properties. Novel magnetoresistive, luminescent and ferroelectric materials have been identified using this approach. These experiments have enhanced our ability to mine the periodic table for new materials with novel properties. In other projects in the materials science area, we have used biomolecules to control the three dimensional structures of nanoclusters in order to further explore this novel form of matter.
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