Publications by authors named "Peter G Schultz"

386 Publications

A synthetic 5,3-cross-link in the cell wall of rod-shaped Gram-positive bacteria.

Proc Natl Acad Sci U S A 2021 Mar;118(11)

Department of Chemistry, Scripps Research, La Jolla, CA 92037;

Gram-positive bacteria assemble a multilayered cell wall that provides tensile strength to the cell. The cell wall is composed of glycan strands cross-linked by nonribosomally synthesized peptide stems. Herein, we modify the peptide stems of the Gram-positive bacterium with noncanonical electrophilic d-amino acids, which when in proximity to adjacent stem peptides form novel covalent 5,3-cross-links. Approximately 20% of canonical cell-wall cross-links can be replaced with synthetic cross-links. While a low level of synthetic cross-link formation does not affect growth and phenotype, at higher levels cell growth is perturbed and bacteria elongate. A comparison of the accumulation of synthetic cross-links over time in Gram-negative and Gram-positive bacteria highlights key differences between them. The ability to perturb cell-wall architecture with synthetic building blocks provides a novel approach to studying the adaptability, elasticity, and porosity of bacterial cell walls.
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http://dx.doi.org/10.1073/pnas.2100137118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980369PMC
March 2021

YAP-dependent proliferation by a small molecule targeting annexin A2.

Nat Chem Biol 2021 Mar 15. Epub 2021 Mar 15.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA.

The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.
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http://dx.doi.org/10.1038/s41589-021-00755-0DOI Listing
March 2021

Discovery and SAR studies of 3-amino-4-(phenylsulfonyl)tetrahydrothiophene 1,1-dioxides as non-electrophilic antioxidant response element (ARE) activators.

Bioorg Chem 2021 Mar 5;108:104614. Epub 2021 Jan 5.

College of Pharmacy, Pusan National University, Busan 46241, Republic of Korea; Department of Chemistry, The Scripps Research Institute, 10550, North Torrey Pines, La Jolla, CA 92037, United States. Electronic address:

The transcription factor NRF2 controls resistance to oxidative insult and is thus a key therapeutic target for treating a number of disease states associated with oxidative stress and aging. We previously reported CBR-470-1, a bis-sulfone which activates NRF2 by increasing the levels of methylglyoxal, a metabolite that covalently modifies NRF2 repressor KEAP1. Here, we report the design, synthesis, and structure activity relationship of a series of bis-sulfones derived from this unexplored chemical template. We identify analogs with sub-micromolar potencies, 7f and 7g, as well as establish that efficacious NRF2 activation can be achieved by non-toxic analogs 7c, 7e, and 9, a key limitation with CBR-470-1. Further efforts to identify non-covalent NRF2 activators of this kind will likely provide new insight into revealing the role of central metabolism in cellular signaling.
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http://dx.doi.org/10.1016/j.bioorg.2020.104614DOI Listing
March 2021

A short ORF-encoded transcriptional regulator.

Proc Natl Acad Sci U S A 2021 Jan;118(4)

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.
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http://dx.doi.org/10.1073/pnas.2021943118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848545PMC
January 2021

Special issue preface.

Authors:
Peter G Schultz

Bioorg Med Chem 2020 12 7;28(24):115814. Epub 2020 Oct 7.

Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, United States. Electronic address:

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http://dx.doi.org/10.1016/j.bmc.2020.115814DOI Listing
December 2020

An orthogonal seryl-tRNA synthetase/tRNA pair for noncanonical amino acid mutagenesis in Escherichia coli.

Bioorg Med Chem 2020 10 28;28(20):115662. Epub 2020 Jul 28.

Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, United States. Electronic address:

We report the development of the orthogonal amber-suppressor pair Archaeoglobus fulgidus seryl-tRNA (Af-tRNA)/Methanosarcina mazei seryl-tRNA synthetase (MmSerRS) in Escherichia coli. Furthermore, the crystal structure of MmSerRS was solved at 1.45 Å resolution, which should enable structure-guided engineering of its active site to genetically encode small, polar noncanonical amino acids (ncAAs).
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http://dx.doi.org/10.1016/j.bmc.2020.115662DOI Listing
October 2020

Engineering of a Potent, Long-Acting NPY2R Agonist for Combination with a GLP-1R Agonist as a Multi-Hormonal Treatment for Obesity.

J Med Chem 2020 09 26;63(17):9660-9671. Epub 2020 Aug 26.

The Scripps Research Institute, d/b/a Calibr, a division of Scripps Research, 11119 North Torrey Pines Road, Suite 100, La Jolla, California 92037, United States.

Bariatric surgery results in increased intestinal secretion of hormones GLP-1 and anorexigenic PYY, which is believed to contribute to the clinical efficacy associated with the procedure. This observation raises the question whether combination treatment with gut hormone analogs might recapitulate the efficacy and mitigate the significant risks associated with surgery. Despite PYY demonstrating excellent efficacy and safety profiles with regard to food intake reduction, weight loss, and glucose control in preclinical animal models, PYY-based therapeutic development remains challenging given a low serum stability and half-life for the native peptide. Here, combined peptide stapling and PEG-fatty acid conjugation affords potent PYY analogs with >14 h rat half-lives, which are expected to translate into a human half-life suitable for once-weekly dosing. Excellent efficacy in glucose control, food intake reduction, and weight loss for lead candidate in combination with our previously reported long-acting GLP-1 analog is demonstrated in a diet-induced obesity mouse model.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00740DOI Listing
September 2020

Antitumor activity of a systemic STING-activating non-nucleotide cGAMP mimetic.

Science 2020 Aug;369(6506):993-999

Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8 T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.
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http://dx.doi.org/10.1126/science.abb4255DOI Listing
August 2020

Discovery of SARS-CoV-2 antiviral drugs through large-scale compound repurposing.

Nature 2020 10 24;586(7827):113-119. Epub 2020 Jul 24.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19). The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.
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http://dx.doi.org/10.1038/s41586-020-2577-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7603405PMC
October 2020

A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals.

bioRxiv 2020 Apr 17. Epub 2020 Apr 17.

The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.
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http://dx.doi.org/10.1101/2020.04.16.044016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263415PMC
April 2020

Semisynthesis of a Bacterium with Non-canonical Cell-Wall Cross-Links.

J Am Chem Soc 2020 06 12;142(25):10910-10913. Epub 2020 Jun 12.

Department of Chemistry, Scripps Research, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

The cell wall is an elaborate framework of peptidoglycan that serves to protect the bacterium against osmotic challenge. This exoskeleton is composed of repeating saccharides covalently cross-linked by peptide stems. The general structure of the cell wall is widely conserved across diverse Gram-negative bacteria. To begin to explore the biological consequence of introducing non-canonical cross-links into the cell wall of , we generated a bacterium where up to 31% of the cell-wall cross-links are formed by a non-enzymatic reaction between a sulfonyl fluoride and an amino group. Bacteria with these non-canonical cell-wall cross-links achieve a high optical density in culture, divide and elongate successfully, and display no loss of outer membrane integrity. This work represents a first step in the design of bacteria with non-canonical "synthetic" cell walls.
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http://dx.doi.org/10.1021/jacs.0c02956DOI Listing
June 2020

2-Sulfonylpyridines as Tunable, Cysteine-Reactive Electrophiles.

J Am Chem Soc 2020 05 29;142(19):8972-8979. Epub 2020 Apr 29.

Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States.

The emerging use of covalent ligands as chemical probes and drugs would benefit from an expanded repertoire of cysteine-reactive electrophiles for efficient and diverse targeting of the proteome. Here we use the endogenous electrophile sensor of mammalian cells, the KEAP1-NRF2 pathway, to discover cysteine-reactive electrophilic fragments from a reporter-based screen for NRF2 activation. This strategy identified a series of 2-sulfonylpyridines that selectively react with biological thiols via nucleophilic aromatic substitution (SAr). By tuning the electrophilicity and appended recognition elements, we demonstrate the potential of the 2-sulfonylpyridine reactive group with the discovery of a selective covalent modifier of adenosine deaminase (ADA). Targeting a cysteine distal to the active site, this molecule attenuates the enzymatic activity of ADA and inhibits proliferation of lymphocytic cells. This study introduces a modular and tunable SAr-based reactive group for targeting reactive cysteines in the human proteome and illustrates the pharmacological utility of this electrophilic series.
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http://dx.doi.org/10.1021/jacs.0c02721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365253PMC
May 2020

Expanding the genetic code of the human hematopoietic system.

Proc Natl Acad Sci U S A 2020 04 6;117(16):8845-8849. Epub 2020 Apr 6.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

The genetic incorporation of noncanonical amino acids (ncAAs) into proteins has been realized in bacteria, yeast, and mammalian cells, and recently, in multicellular organisms including plants and animals. However, the addition of new building blocks to the genetic code of tissues from human origin has not yet been achieved. To this end, we report a self-replicating Epstein-Barr virus-based episomal vector for the long-term encoding of ncAAs in human hematopoietic stem cells and reconstitution of this genetically engineered hematopoietic system in mice.
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http://dx.doi.org/10.1073/pnas.1914408117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7183198PMC
April 2020

New Generation Oxyntomodulin Peptides with Improved Pharmacokinetic Profiles Exhibit Weight Reducing and Anti-Steatotic Properties in Mice.

Bioconjug Chem 2020 04 3;31(4):1167-1176. Epub 2020 Apr 3.

Calibr at The Scripps Research Institute, La Jolla, California 92037, United States.

Oxyntomodulin (OXM) is an intestinal peptide hormone that activates both glucagon-like peptide-1 (GLP-1) and glucagon (GCG) receptors. The natural peptide reduces body weight in obese subjects and exhibits direct acute glucoregulatory effects in patients with type II diabetes. However, the clinical utility of OXM is limited due to its lower potency and short half-life. To overcome these issues, we developed stapled, long-acting, and highly potent OXM analogs with balanced activities at both GLP-1 and GCG receptors. The lead molecule exhibits potent and long-lasting effects on glucose control, body weight loss, and reduction of hepatic fat reduction in DIO mice. Importantly, significantly reversed hepatic steatosis; reduced liver weight, total cholesterol, and hepatic triglycerides; and improved markers of liver function in a nonalcoholic steatohepatitis (NASH) mouse model. A symmetrical version of the peptide was also shown to be more efficacious and long-lasting in controlling glucose than semaglutide and the clinical candidate cotadutide in wild-type mice, highlighting the utility of our designs of the dual agonist as a potential new therapy for diabetes and liver diseases.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00093DOI Listing
April 2020

Neratinib protects pancreatic beta cells in diabetes.

Nat Commun 2019 11 1;10(1):5015. Epub 2019 Nov 1.

Centre for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany.

The loss of functional insulin-producing β-cells is a hallmark of diabetes. Mammalian sterile 20-like kinase 1 (MST1) is a key regulator of pancreatic β-cell death and dysfunction; its deficiency restores functional β-cells and normoglycemia. The identification of MST1 inhibitors represents a promising approach for a β-cell-protective diabetes therapy. Here, we identify neratinib, an FDA-approved drug targeting HER2/EGFR dual kinases, as a potent MST1 inhibitor, which improves β-cell survival under multiple diabetogenic conditions in human islets and INS-1E cells. In a pre-clinical study, neratinib attenuates hyperglycemia and improves β-cell function, survival and β-cell mass in type 1 (streptozotocin) and type 2 (obese Lepr) diabetic mouse models. In summary, neratinib is a previously unrecognized inhibitor of MST1 and represents a potential β-cell-protective drug with proof-of-concept in vitro in human islets and in vivo in rodent models of both type 1 and type 2 diabetes.
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http://dx.doi.org/10.1038/s41467-019-12880-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825211PMC
November 2019

Small-Molecule Stimulators of NRF1 Transcriptional Activity.

Chembiochem 2020 07 8;21(13):1816-1819. Epub 2019 Nov 8.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037, USA.

The transcription factor nuclear factor erythroid 2-related factor 1 (NRF1) maintains proteostasis and promotes cellular resilience by stimulating the transcription of proteasomal subunits and a host of protective enzymes. Although NRF1 activation would likely be beneficial in a number of disease states, information regarding its ligandability and upstream regulation are lacking. Herein we report a high-throughput chemical screen that identified selective stimulators of NRF1-driven transcription, including unannotated inhibitors of the ubiquitin proteasome system (UPS) as well as two non-UPS-targeted compounds that synergistically activate NRF1 in the context of submaximal UPS inhibition. This work introduces a suite of tool molecules to study the NRF1 transcriptional response and to uncover the druggable components governing NRF1 activity in cells.
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http://dx.doi.org/10.1002/cbic.201900487DOI Listing
July 2020

A General Strategy for Engineering Noncanonical Amino Acid Dependent Bacterial Growth.

J Am Chem Soc 2019 10 4;141(41):16213-16216. Epub 2019 Oct 4.

Department of Chemistry and Skaggs Institute for Chemical Biology , The Scripps Research Institute , 10550 North Torrey Pines Road , La Jolla , California 92037 , United States.

Synthetic auxotrophy in which bacterial viability depends on the presence of a synthetic amino acid provides a robust strategy for the containment of genetically modified organisms and the development of safe, live vaccines. However, a simple, general strategy to evolve essential proteins to be dependent on synthetic amino acids is lacking. Using a temperature-sensitive selection system, we evolved an () sliding clamp variant with an orthogonal protein-protein interface, which contains a Leu273 to -benzoylphenyl alanine (pBzF) mutation. The strain with this variant DNA clamp has a very low escape frequency (<10), and its growth is strictly dependent on the presence of pBzF. This selection strategy can be generally applied to create ncAA dependence of other organisms with DNA clamp homologues.
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http://dx.doi.org/10.1021/jacs.9b08491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7501724PMC
October 2019

Toward a Synthetic Yeast Endosymbiont with a Minimal Genome.

J Am Chem Soc 2019 09 26;141(35):13799-13802. Epub 2019 Aug 26.

The Department of Chemistry , Scripps Research , La Jolla , California 92037 , United States.

Based on the endosymbiotic theory, one of the key events that occurred during mitochondrial evolution was an extensive loss of nonessential genes from the protomitochondrial endosymbiont genome and transfer of some of the essential endosymbiont genes to the host nucleus. We have developed an approach to recapitulate various aspects of endosymbiont genome minimization using a synthetic system consisting of endosymbionts within host yeast cells. As a first step, we identified a number of auxotrophs of central metabolites that can form viable endosymbionts within yeast cells. These studies provide a platform to identify nonessential biosynthetic pathways that can be deleted in the endosymbionts to investigate the evolutionary adaptations in the host and endosymbiont during the evolution of mitochondria.
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http://dx.doi.org/10.1021/jacs.9b08290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999831PMC
September 2019

A tumor-targeted immune checkpoint blocker.

Proc Natl Acad Sci U S A 2019 08 22;116(32):15889-15894. Epub 2019 Jul 22.

Key Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, 100101 Beijing, China;

To direct checkpoint inhibition to the tumor microenvironment, while avoiding systemic immune activation, we have synthesized a bispecific antibody [norleucine4, d-Phe7]-melanocyte stimulating hormone (NDP-MSH)-antiprogrammed cell death-ligand 1 antibody (αPD-L1) by conjugating a melanocyte stimulating hormone (α-MSH) analog to the antiprogrammed cell death-ligand 1 to (αPD-L1) antibody avelumab. This bispecific antibody can bind to both the melanocortin-1 receptor (MC1R) and to PD-L1 expressed on melanoma cells and shows enhanced specific antitumor efficacy in a syngeneic B16-SIY melanoma mouse model compared with the parental antibody at a 5 mg/kg dose. Moreover, the bispecific antibody showed increased infiltrated T cells in the tumor microenvironment. These results suggest that a tumor-targeted PD-L1-blocking bispecific antibody could have a therapeutic advantage in vivo, especially when used in combination with other checkpoint inhibitors.
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http://dx.doi.org/10.1073/pnas.1905646116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689898PMC
August 2019

Site-Specific Incorporation of a Dithiolane Containing Amino Acid into Proteins.

Bioconjug Chem 2019 08 23;30(8):2102-2105. Epub 2019 Jul 23.

Department of Chemistry and Skaggs Institute for Chemical Biology , The Scripps Research Institute , 10550 N. Torrey Pines Road , La Jolla , California 92037 , United States.

We have genetically encoded a dithiolane containing amino acid (dtF) in () using a polyspecific aminoacyl-tRNA synthetase (aaRS)/amber suppressor tRNA pair. To demonstrate the utility of dtF for bioapplications, we synthesized gold nanoparticle (AuNP) constructs with a mutant superfolder green fluorescent protein (sfGFP) [sfGFP-AuNP] as a model for the protein-metal conjugation. The resulting sfGFP-AuNP constructs show directional homogeneity and enhanced chemical durability compared to their cysteine analogues toward excess environmental 1,4-dithiothreitol (DTT).
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00413DOI Listing
August 2019

A Single Reactive Noncanonical Amino Acid Is Able to Dramatically Stabilize Protein Structure.

ACS Chem Biol 2019 06 4;14(6):1150-1153. Epub 2019 Jun 4.

Department of Chemistry and Skaggs Institute for Chemical Biology , The Scripps Research Institute , La Jolla , California 92037 , United States.

A p-isothiocyanate phenylalanine mutant of the homodimeric protein homoserine o-succinyltransferase (MetA) was isolated in a temperature dependent selection from a library of metA mutants containing noncanonical amino acids. This mutant protein has a dramatic increase of 24 °C in thermal stability compared to the wild type protein. Peptide mapping experiments revealed that the isothiocyanate group forms a thiourea cross-link to the N terminal proline of the other monomer, despite the two positions being >30 Å apart in the X-ray crystal structure of the wild type protein. These results show that an expanded set of building blocks beyond the canonical 20 amino acids can lead to significant changes in the properties of proteins.
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http://dx.doi.org/10.1021/acschembio.9b00002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791372PMC
June 2019

Discovery of short-course antiwolbachial quinazolines for elimination of filarial worm infections.

Sci Transl Med 2019 05;11(491)

Research Foundation in Tropical Diseases and the Environment, Buea, Cameroon.

Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets , a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of in the in vivo filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify elimination in filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in , , and in vivo preclinical models of filarial disease and in vitro selectivity against (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.
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http://dx.doi.org/10.1126/scitranslmed.aav3523DOI Listing
May 2019

A cell type-selective apoptosis-inducing small molecule for the treatment of brain cancer.

Proc Natl Acad Sci U S A 2019 03 7;116(13):6435-6440. Epub 2019 Mar 7.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

Glioblastoma multiforme (GBM; grade IV astrocytoma) is the most prevalent and aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation, tumor maintenance, metastasis, drug resistance, and recurrence following surgery. Here we report the identification of a small molecule, termed RIPGBM, from a cell-based chemical screen that selectively induces apoptosis in multiple primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of this compound appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an orthotopic intracranial GBM CSC tumor xenograft mouse model, RIPGBM was found to significantly suppress tumor formation in vivo. Our chemical genetics-based approach has identified a drug candidate and a potential drug target that provide an approach to the development of treatments for this devastating disease.
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http://dx.doi.org/10.1073/pnas.1816626116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442583PMC
March 2019

Enhancing Protein Stability with Genetically Encoded Noncanonical Amino Acids.

J Am Chem Soc 2018 11 15;140(47):15997-16000. Epub 2018 Nov 15.

Department of Chemistry and Skaggs Institute for Chemical Biology , The Scripps Research Institute , La Jolla , California 92037 , United States.

The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins with new or enhanced properties using a larger set of building blocks. To this end, we have been able to select mutant proteins with enhanced thermal properties from a library of E. coli homoserine O-succinyltransferase ( metA) mutants containing randomly incorporated noncanonical amino acids. Here, we show that substitution of Phe 21 with ( p-benzoylphenyl)alanine (pBzF), increases the melting temperature of E. coli metA by 21 °C. This dramatic increase in thermal stability, arising from a single mutation, likely results from a covalent adduct between Cys 90 and the keto group of pBzF that stabilizes the dimeric form of the enzyme. These experiments show that an expanded genetic code can provide unique solutions to the evolution of proteins with enhanced properties.
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http://dx.doi.org/10.1021/jacs.8b07157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426444PMC
November 2018

Visualization of the Delivery and Release of Small RNAs Using Genetic Code Expansion and Unnatural RNA-Binding Proteins.

Bioconjug Chem 2018 12 13;29(12):3982-3986. Epub 2018 Nov 13.

Department of Chemistry and Biomolecular Sciences , University of Ottawa , 10 Marie Curie Pvt. , Ottawa , Ontario K1N 6N5 , Canada.

Endogenously expressed noncoding RNAs are regulators of mRNA translation and affect diverse biological pathways spanning embryogenesis to cholesterol and fatty acid metabolism. Recently, microRNAs have become an important therapeutic target with strategies that employ oligonucleotides as both mimics and inhibitors of target microRNAs, successfully altering gene expression and cellular pathways in relevant contexts. However, delivery of these exogenous effectors remains a major challenge. Here, we present a method for evaluating noncoding RNA delivery using the viral suppressor of RNA silencing (VSRS) protein p19, optimized for cellular delivery of small RNAs. Using genetic code expansion technology, p-azidophenylalanine (AzF) was incorporated into a recombinant p19 protein and used to develop a fluorescence resonance energy transfer (FRET) sensor. AzF was used to attach FRET acceptor moieties using bioorthogonal chemistry. We show that this strategy not only gives rise to FRET signals that report on small RNA binding, but also allows for fluorescence quenching as well, convenient for measuring RNA release. We demonstrate the successful use of a modified version of the probe to track the delivery and release of small RNAs into mammalian cells. The results provide a basis for a further development of vehicles for small RNA delivery and release for intervening in noncoding RNA biology.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00649DOI Listing
December 2018

Engineering yeast endosymbionts as a step toward the evolution of mitochondria.

Proc Natl Acad Sci U S A 2018 11 29;115(46):11796-11801. Epub 2018 Oct 29.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in an archaeal host cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between and An ADP/ATP translocase-expressing provided ATP to a respiration-deficient yeast mutant and enabled growth of a yeast- chimera on a nonfermentable carbon source. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic thiamin auxotroph. Expression of several SNARE-like proteins in was also required, likely to block lysosomal degradation of intracellular bacteria. This chimeric system was stable for more than 40 doublings, and GFP-expressing endosymbionts could be observed in the yeast by fluorescence microscopy and X-ray tomography. This readily manipulated system should allow experimental delineation of host-endosymbiont adaptations that occurred during evolution of the current, highly reduced mitochondrial genome.
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http://dx.doi.org/10.1073/pnas.1813143115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243291PMC
November 2018

A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Nature 2018 10 15;562(7728):600-604. Epub 2018 Oct 15.

Department of Chemistry, University of Chicago, Chicago, IL, USA.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes, and regulate cellular functions such as metabolism and transcription. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF2, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases.
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http://dx.doi.org/10.1038/s41586-018-0622-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444936PMC
October 2018

The ReFRAME library as a comprehensive drug repurposing library and its application to the treatment of cryptosporidiosis.

Proc Natl Acad Sci U S A 2018 10 3;115(42):10750-10755. Epub 2018 Oct 3.

California Institute for Biomedical Research, La Jolla, CA 92037;

The chemical diversity and known safety profiles of drugs previously tested in humans make them a valuable set of compounds to explore potential therapeutic utility in indications outside those originally targeted, especially neglected tropical diseases. This practice of "drug repurposing" has become commonplace in academic and other nonprofit drug-discovery efforts, with the appeal that significantly less time and resources are required to advance a candidate into the clinic. Here, we report a comprehensive open-access, drug repositioning screening set of 12,000 compounds (termed ReFRAME; Repurposing, Focused Rescue, and Accelerated Medchem) that was assembled by combining three widely used commercial drug competitive intelligence databases (Clarivate Integrity, GVK Excelra GoStar, and Citeline Pharmaprojects), together with extensive patent mining of small molecules that have been dosed in humans. To date, 12,000 compounds (∼80% of compounds identified from data mining) have been purchased or synthesized and subsequently plated for screening. To exemplify its utility, this collection was screened against spp., a major cause of childhood diarrhea in the developing world, and two active compounds previously tested in humans for other therapeutic indications were identified. Both compounds, VB-201 and a structurally related analog of ASP-7962, were subsequently shown to be efficacious in animal models of infection at clinically relevant doses, based on available human doses. In addition, an open-access data portal (https://reframedb.org) has been developed to share ReFRAME screen hits to encourage additional follow-up and maximize the impact of the ReFRAME screening collection.
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http://dx.doi.org/10.1073/pnas.1810137115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6196526PMC
October 2018

Bacterial Genome Containing Chimeric DNA-RNA Sequences.

J Am Chem Soc 2018 09 30;140(36):11464-11473. Epub 2018 Aug 30.

The Scripps Research Institute , 10550 North Torrey Pines Road , La Jolla , California 92037 , United States.

Almost five decades ago Crick, Orgel, and others proposed the RNA world hypothesis. Subsequent studies have raised the possibility that RNA might be able to support both genotype and phenotype, and the function of RNA templates has been studied in terms of evolution, replication, and catalysis. Recently, we engineered strains of E. coli in which a large fraction of 2'-deoxycytidine in the genome is substituted with the modified base 5-hydroxymethyl-2'-deoxycytidine. We now report the generation of mutant strains derived from these engineered bacteria that show significant (∼40-50%) ribonucleotide content in their genome. We have begun to characterize the properties of these chimeric genomes and the corresponding strains to determine the circumstances under which E. coli can incorporate ribonucleotides into its genome and herein report our initial observations.
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http://dx.doi.org/10.1021/jacs.8b07046DOI Listing
September 2018