Peter G Schultz

Publications (287)2528.39 Total impact

• Article: An Expanded Genetic Code In Mammalian Cells With A Functional Quadruplet Codon.

  Wei Niu, Peter G Schultz, Jiantao Guo
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  ABSTRACT: We have utilized in vitro evolution to identify tRNA variants with significantly enhanced activity for the incorporation of unnatural amino acids into proteins in response to a quadruplet codon in both bacterial and mammalian cells. This approach will facilitate the creation of an optimized and standardized system for the genetic incorporation of unnatural amino acids using quadruplet codons, which will allow the biosynthesis of biopolymers that contain multiple unnatural building blocks.
  ACS Chemical Biology 05/2013; · 6.45 Impact Factor
• Article: Protein conjugation with genetically encoded unnatural amino acids.

  Chan Hyuk Kim, Jun Y Axup, Peter G Schultz
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  ABSTRACT: The site-specific incorporation of unnatural amino acids with orthogonal chemical reactivity into proteins enables the synthesis of structurally defined protein conjugates. Amino acids containing ketone, azide, alkyne, alkene, and tetrazine side chains can be genetically encoded in response to nonsense and frameshift codons. These bio-orthogonal chemical handles allow precise control over the site and stoichiometry of conjugation, and have enabled medicinal chemistry-like optimization of the physical and biological properties of protein conjugates, especially the next-generation protein therapeutics.
  Current opinion in chemical biology 05/2013; · 8.30 Impact Factor
• Article: Small molecule mediated proliferation of primary retinal pigment epithelial cells.

  Jonathan G Swoboda, Jimmy Elliott, Vishal Deshmukh, Lorenzo de Lichtervelde, Weijun Shen, Matthew S Tremblay, Eric C Peters, Charles Y Cho, Bin Lu, Sergey Girman, Shaomei Wang, Peter G Schultz
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  ABSTRACT: Retinal pigment epithelial (RPE) cells form a monolayer adjacent to the retina and play a critical role in the visual light cycle. Degeneration of this layer results in vision loss, causing retinal disorders such as age-related macular degeneration. Cell transplant therapies exist to restore vision loss; however, risks associated with and an inadequate supply of donor cells have limited their therapeutic success. The identification of factors that proliferate RPE cells ex vivo could provide a renewable source of cells for the treatment of such disorders. We show that a small molecule (WS3) can reversibly proliferate primary RPE cells isolated from fetal and adult human donors. Following withdrawal of WS3, RPE cells differentiate into a functional monolayer, as exhibited by their expression of mature RPE genes and phagocytosis of photoreceptor outer segments. Furthermore, chemically expanded RPE cells preserve vision when transplanted into dystrophic Royal College of Surgeons (RCS) rats, a well-established model of retinal degeneration.
  ACS Chemical Biology 04/2013; · 6.45 Impact Factor
• Article: A Tryptophanyl-tRNA Synthetase/tRNA Pair for Unnatural Amino Acid Mutagenesis in E. coli.

  Abhishek Chatterjee, Han Xiao, Peng-Yu Yang, Gautam Soundararajan, Peter G Schultz
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  ABSTRACT: Selected: A tryptophanyl-tRNA synthetase(TrpRS)/tRNA pair was evolved to genetically encode tryptophan analogues and other unnatural amino acids with large side chains in E. coli. A selection scheme was employed to identify TrpRS variants able to selectively charge tRNACUA (Trp) with Trp analogues. Substitution of Trp66 in enhanced cyan fluorescent protein (ECFP) with these Trp analogues afforded ECFP variants with novel spectral properties.
  Angewandte Chemie International Edition 04/2013; · 13.45 Impact Factor
• Article: Identification of metal ion binding peptides containing unnatural amino acids by phage display.

  Jonathan W Day, Chan Hyuk Kim, Vaughn V Smider, Peter G Schultz
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  ABSTRACT: The bidentate metal binding amino acid bipyridylalanine (BpyAla) was incorporated into a disulfide linked cyclic peptide phage displayed library to identify metal ion binding peptides. Selection against Ni(2+)-nitrilotriacetic acid (NTA) enriched for sequences containing histidine and BpyAla. BpyAla predominated when selections were carried out at lower pH, consistent with the differential pKa's of histidine and BpyAla. Two peptides containing BpyAla were synthesized and found to bind Ni(2+) with low micromolar dissociation constants. Incorporation of BpyAla and other metal binding amino acids into peptide and protein libraries should enable the evolution of novel binding and catalytic activities.
  Bioorganic & medicinal chemistry letters 03/2013; · 2.65 Impact Factor
• Article: Eupalinilide E inhibits erythropoiesis and promotes the expansion of hematopoietic progenitor cells.

  Lorenzo de Lichtervelde, Anthony E Boitano, Ying Wang, Philipp Krastel, Frank Petersen, Michael P Cooke, Peter G Schultz
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  ABSTRACT: Hematopoietic stem cells (HSCs) are the progenitor cells that give rise to all blood cells. The ability to control HSC differentiation has the potential to improve the success of bone marrow transplants and the production of functional blood cells ex vivo. Here we performed an unbiased screen using primary human CD34+ hematopoietic stem and progenitor cells (HSPCs) to identify natural products that selectively control their differentiation. We identified a plant-derived natural product, eupalinilide E, that promotes the ex-vivo expansion of HSPCs and hinders the in vitro development of erythrocytes. This activity was additive with aryl hydrocarbon receptor (AhR) antagonists, which are also known to expand HSCs and currently in clinical development. These findings reveal a new activity for eupalinilide E, and suggest that it may be a useful tool to probe the mechanisms of hematopoiesis and improve the ex vivo production of progenitors for therapeutic purposes.
  ACS Chemical Biology 02/2013; · 6.45 Impact Factor
• Article: Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation.

  Feng Wang, Shiladitya Sen, Yong Zhang, Insha Ahmad, Xueyong Zhu, Ian A Wilson, Vaughn V Smider, Thomas J Magliery, Peter G Schultz
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  ABSTRACT: Somatic hypermutation and clonal selection lead to B cells expressing high-affinity antibodies. Here we show that somatic mutations not only play a critical role in antigen binding, they also affect the thermodynamic stability of the antibody molecule. Somatic mutations directly involved in antigen recognition by antibody 93F3, which binds a relatively small hapten, reduce the melting temperature compared with its germ-line precursor by up to 9 °C. The destabilizing effects of these mutations are compensated by additional somatic mutations located on surface loops distal to the antigen binding site. Similarly, somatic mutations enhance both the affinity and thermodynamic stability of antibody OKT3, which binds the large protein antigen CD3. Analysis of the crystal structures of 93F3 and OKT3 indicates that these somatic mutations modulate antibody stability primarily through the interface of the heavy and light chain variable domains. The historical view of antibody maturation has been that somatic hypermutation and subsequent clonal selection increase antigen-antibody specificity and binding energy. Our results suggest that this process also optimizes protein stability, and that many peripheral mutations that were considered to be neutral are required to offset deleterious effects of mutations that increase affinity. Thus, the immunological evolution of antibodies recapitulates on a much shorter timescale the natural evolution of enzymes in which function and thermodynamic stability are simultaneously enhanced through mutation and selection.
  Proceedings of the National Academy of Sciences 02/2013; · 9.68 Impact Factor
• Article: A versatile platform for single and multiple unnatural amino acid mutagenesis in Escherichia coli.

  Abhishek Chatterjee, Sophie B Sun, Jennifer Lynn Furman, Han Xiao, Peter G Schultz
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  ABSTRACT: To site-specifically incorporate an unnatural amino acid (UAA) into target proteins in Escherichia coli a suppressor plasmid is used, which provides an engineered suppressor tRNA and an aminoacyl-tRNA synthetase (aaRS) specific for the UAA of interest. The continuous drive to further improve UAA incorporation efficiency in E. coli has resulted in several generations of suppressor plasmids. Here we describe a new, highly efficient suppressor plasmid, pUltra, harboring a single copy each of the tRNA and aaRS expression cassettes that exhibits higher suppression activity relative to its predecessors. This system is able to efficiently incorporate up to three UAAs within the same protein at levels up to 30% of wild-type protein expression. Its unique origin of replication (CloDF13) and antibiotic resistance marker (spectinomycin), allows pUltra to be used in conjunction with the previously reported pEVOL suppressor plasmid, each encoding a distinct tRNA/aaRS pair, to simultaneously insert two different UAAs into the same protein. We demonstrate the utility of this system by efficiently incorporating two bio-orthogonal UAAs containing keto and azido side chains into ketosteroid isomerase and subsequently derivatizing these amino acids with two distinct fluorophores, capable of FRET interaction. Finally, owing to its minimal composition, two different tRNA/aaRS pairs were encoded in pUltra, allowing the generation of a single plasmid capable of dual suppression. The high suppression efficiency and the ability to harbor multiple tRNA/aaRS pairs make pUltra a useful system to carry out single and multiple UAA mutagenesis in E. coli.
  Biochemistry 02/2013; · 3.42 Impact Factor
• Article: A Small molecule inducer of β cell proliferation identified by high-throughput screening.

  Weijun Shen, Matthew S Tremblay, Vishal A Deshmukh, Weidong Wang, Christophe M Filippi, George Harb, You-Qing Zhang, Anwesh Kamireddy, Janine E Baaten, Qihui Jin, Tom Wu, Jonathan G Swoboda, Charles Y Cho, Jing Li, Bryan A Laffitte, Peter McNamara, Richard Glynne, Xu Wu, Ann E Herman, Peter G Schultz
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  ABSTRACT: The identification of factors that promote β cell proliferation could ultimately move type 1 diabetes treatment away from insulin injection therapy and towards a cure. We have performed high-throughput, cell-based screens using rodent β cell lines to identify molecules that induce proliferation of β cells. Herein we report the discovery and characterization of WS6, a novel small molecule that promotes β cell proliferation in rodent and human primary islets. In the RIP-DTA mouse model of β cell ablation, WS6 normalized blood glucose and induced concomitant increases in β cell proliferation and β cell number. Affinity pulldown and kinase profiling studies implicate Erb3 binding protein (EBP)-1 and the IκB kinase (IKK) pathway in the mechanism of action of WS6.
  Journal of the American Chemical Society 01/2013; · 9.91 Impact Factor
• Source

  Available from: Costas A Lyssiotis

  Article: Small molecule-based approaches to adult stem cell therapies.

  Luke L Lairson, Costas A Lyssiotis, Shoutian Zhu, Peter G Schultz
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  ABSTRACT: There is considerable interest in the development of stem cell-based strategies for the treatment of a broad range of human diseases, including neurodegenerative, autoimmune, cardiovascular, and musculoskeletal diseases. To date, such regenerative approaches have focused largely on the development of cell transplantation therapies using cells derived from pluripotent embryonic stem cells (ESCs). Although there have been exciting preliminary reports describing the efficacy of ESC-derived replacement therapies, approaches involving ex vivo manipulated ESCs are hindered by issues of mutation, immune rejection, and ethical controversy. An alternative approach involves direct in vivo modulation or ex vivo expansion of endogenous adult stem cell populations using drug-like small molecules. Here we describe chemical approaches to the regulation of somatic stem cell biology that are yielding new biological insights and that may ultimately lead to innovative new medicines.
  Annual Review of Pharmacology 01/2013; 53:107-25. · 21.64 Impact Factor
• Article: Self-Assembled Antibody Multimers through Peptide Nucleic Acid Conjugation.

  Stephanie A Kazane, Jun Y Axup, Chan Hyuk Kim, Mihai Ciobanu, Erik D Wold, Sofia Barluenga, Benjamin A Hutchins, Peter G Schultz, Nicolas Winssinger, Vaughn V Smider
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  ABSTRACT: With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody-oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency and geometry. Using this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and in vitro characterization of antibody constructs with unique specificities and molecular architectures.
  Journal of the American Chemical Society 12/2012; · 9.91 Impact Factor
• Source

  Available from: Antonio Santidrian

  Article: Synthesis of site-specific antibody-drug conjugates using unnatural amino acids.

  Jun Y Axup, Krishna M Bajjuri, Melissa Ritland, Benjamin M Hutchins, Chan Hyuk Kim, Stephanie A Kazane, Rajkumar Halder, Jane S Forsyth, Antonio F Santidrian, Karin Stafin, [......], Hon Tran, Aaron J Seller, Sandra L Biroc, Aga Szydlik, Jason K Pinkstaff, Feng Tian, Subhash C Sinha, Brunhilde Felding-Habermann, Vaughn V Smider, Peter G Schultz
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  ABSTRACT: Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2(+) cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.
  Proceedings of the National Academy of Sciences 09/2012; 109(40):16101-6. · 9.68 Impact Factor
• Article: Unnatural amino Acid mutagenesis of fluorescent proteins.

  Feng Wang, Wei Niu, Jiantao Guo, Peter G Schultz
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  ABSTRACT: Tyrosine 66 of a green fluorescent protein (GFP) was substituted with unnatural amino acids carrying boronate, azido, nitro, and keto substituents. In general, the ${{\lambda _{{\rm{{\rm em}}}}^{{\rm{{\rm max}}}} }}$values of these GFP mutants is blue-shifted relative to that of GFP, and the fluorescence intensity of the boronate variant increases upon oxidation. The X-ray crystal structures of the keto and boronate GFP mutants provide explanations of their altered fluorescence properties.
  Angewandte Chemie International Edition 09/2012; 51(40):10132-5. · 13.45 Impact Factor
• Article: Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli.

  Abhishek Chatterjee, Han Xiao, Peter G Schultz
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  ABSTRACT: The site-specific incorporation of unnatural amino acids (UAAs) into proteins in living cells relies on an engineered tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair, orthogonal to the host cell, to deliver the UAA of choice in response to a unique nonsense or frameshift codon. Here we report the generation of mutually orthogonal prolyl-tRNA/prolyl-tRNA synthase (ProRS) pairs derived from an archaebacterial ancestor for use in Escherichia coli. By reprogramming the anticodon-binding pocket of Pyrococcus horikoshii ProRS (PhProRS), we were able to identify synthetase variants that recognize engineered Archaeoglobus fulgidus prolyl-tRNAs (Af-tRNA(Pro)) with three different anticodons: CUA, AGGG, and CUAG. Several of these evolved PhProRSs show specificity toward a particular anticodon variant of Af-tRNA(Pro), whereas others are promiscuous. Further evolution of the Af-tRNA(Pro) led to a variant exhibiting significantly improved amber suppression efficiency. Availability of a prolyl-tRNA/aaRS pair should enable site-specific incorporation of proline analogs and other N-modified UAAs into proteins in E. coli. The evolution of mutually orthogonal prolyl-tRNA/ProRS pairs demonstrates the plasticity of the tRNA-aaRS interface and should facilitate the incorporation of multiple, distinct UAAs into proteins.
  Proceedings of the National Academy of Sciences 08/2012; 109(37):14841-6. · 9.68 Impact Factor
• Article: Euphohelioscopin A is a PKC activator capable of inducing macrophage differentiation.

  Lorenzo de Lichtervelde, Corina E Antal, Anthony E Boitano, Ying Wang, Philipp Krastel, Frank Petersen, Alexandra C Newton, Michael P Cooke, Peter G Schultz
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  ABSTRACT: To identify small molecules that selectively control hematopoietic stem cell differentiation, we performed an unbiased screen using primary human CD34(+) cells. We identified a plant-derived natural product, euphohelioscopin A, capable of selectively differentiating CD34(+) cells down the granulocyte/monocytic lineage. Euphohelioscopin A also inhibits proliferation and induces differentiation of the myeloid leukemia cell lines THP-1 and HL-60. Mechanistic studies revealed that euphohelioscopin A is an activator of protein kinase C (PKC), and that the promonocytic effects of this natural product are mediated by PKC activation. In addition to shedding insights into normal hematopoiesis, this work may ultimately facilitate the application of stem cell therapies to a host of myeloid dysfunctions.
  Chemistry & biology 08/2012; 19(8):994-1000. · 6.52 Impact Factor
• Article: A small molecule promotes mitochondrial fusion in Mammalian cells.

  Danling Wang, Jianing Wang, Ghislain M C Bonamy, Shelly Meeusen, Richard G Brusch, Carolina Turk, Pengyu Yang, Peter G Schultz
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  ABSTRACT: Mitochondrial dynamics: An image-based screen identified a small molecule, M1, that specifically promotes the fusion of fragmented mitochondria and protects cells from mitochondrial-fragmentation-associated cell death. Mechanistic studies revealed that M1 shifts the mitochondrial dynamic balance towards fusion.
  Angewandte Chemie International Edition 08/2012; 51(37):9302-5. · 13.45 Impact Factor
• Source

  Available from: pnas.org

  Article: An image-based screen identifies a small molecule regulator of megakaryopoiesis.

  Anthony E Boitano, Lorenzo de Lichtervelde, Jennifer L Snead, Michael P Cooke, Peter G Schultz
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  ABSTRACT: Molecules that control the lineage commitment of hematopoietic stem cells (HSCs) may allow the expansion of enriched progenitor populations for both research and therapeutic uses. In an effort to better understand and control the differentiation of HSCs to megakaryocytes, we carried out an image-based screen of a library of 50,000 heterocycles using primary human CD34(+) cells. A class of naphthyridinone derivatives was identified that induces the differentiation of common myeloid progenitors (CMP) to megakaryocytes. Kinase profiling and subsequent functional assays revealed that these compounds act through inhibition of platelet-derived growth factor receptor (PDGFR) signaling in CMPs. Such molecules may ultimately have clinical utility in the treatment of thrombocytopenia.
  Proceedings of the National Academy of Sciences 08/2012; 109(35):14019-23. · 9.68 Impact Factor
• Source

  Available from: Steve A Kay

  Article: Identification of small molecule activators of cryptochrome.

  Tsuyoshi Hirota, Jae Wook Lee, Peter C St John, Mariko Sawa, Keiko Iwaisako, Takako Noguchi, Pagkapol Y Pongsawakul, Tim Sonntag, David K Welsh, David A Brenner, Francis J Doyle, Peter G Schultz, Steve A Kay
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  ABSTRACT: Impairment of the circadian clock has been associated with numerous disorders, including metabolic disease. Although small molecules that modulate clock function might offer therapeutic approaches to such diseases, only a few compounds have been identified that selectively target core clock proteins. From an unbiased cell-based circadian phenotypic screen, we identified KL001, a small molecule that specifically interacts with cryptochrome (CRY). KL001 prevented ubiquitin-dependent degradation of CRY, resulting in lengthening of the circadian period. In combination with mathematical modeling, our studies using KL001 revealed that CRY1 and CRY2 share a similar functional role in the period regulation. Furthermore, KL001-mediated CRY stabilization inhibited glucagon-induced gluconeogenesis in primary hepatocytes. KL001 thus provides a tool to study the regulation of CRY-dependent physiology and aid development of clock-based therapeutics of diabetes.
  Science 07/2012; 337(6098):1094-7. · 31.20 Impact Factor
• Article: Site-specific incorporation of ε-N-crotonyllysine into histones.

  Chan Hyuk Kim, Mingchao Kang, Hak Joong Kim, Abhishek Chatterjee, Peter G Schultz
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  ABSTRACT: A novel post-translationally modified amino acid, crotonyllysine (Kcr), was genetically incorporated into proteins in bacterial and mammalian cells using an evolved pyrrolysyl-tRNA/synthetase-tRNA pair. The ability to produce histones with homogenous, site-specific Kcr modifications will be valuable in elucidating the biological role of this recently identified post-translational modification.
  Angewandte Chemie International Edition 06/2012; 51(29):7246-9. · 13.45 Impact Factor
• Article: Synthesis of bispecific antibodies using genetically encoded unnatural amino acids.

  Chan Hyuk Kim, Jun Y Axup, Anna Dubrovska, Stephanie A Kazane, Benjamin A Hutchins, Erik D Wold, Vaughn V Smider, Peter G Schultz
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  ABSTRACT: Bispecific antibodies were constructed using genetically encoded unnatural amino acids with orthogonal chemical reactivity. A two-step process afforded homogeneous products in excellent yield. Using this approach, we synthesized an anti-HER2/anti-CD3 bispecific antibody, which efficiently cross-linked HER2+ cells and CD3+ cells. In vitro effector-cell mediated cytotoxicity was observed at picomolar concentrations.
  Journal of the American Chemical Society 05/2012; 134(24):9918-21. · 9.91 Impact Factor