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1. β-Hairpin Alignment Alters Oligomer Formation in Aβ-Derived Peptides.

Author

Ruttenberg, Sarah, Kreutzer, Adam, Truex, Nicholas, and Nowick, James

Subjects
Humans, Amyloid beta-Peptides, Models, Molecular, Alzheimer Disease, Protein Conformation, Crystallography, X-Ray, Peptide Fragments
Abstract

Amyloid-β (Aβ) forms heterogeneous oligomers, which are implicated in the pathogenesis of Alzheimers disease (AD). Many Aβ oligomers consist of β-hairpin building blocks─Aβ peptides in β-hairpin conformations. β-Hairpins of Aβ can adopt a variety of alignments, but the role that β-hairpin alignment plays in the formation and heterogeneity of Aβ oligomers is poorly understood. To explore the effect of β-hairpin alignment on the oligomerization of Aβ peptides, we designed and studied two model peptides with two different β-hairpin alignments. Peptides Aβm17-36 and Aβm17-35 mimic two different β-hairpins that Aβ can form, the Aβ17-36 and Aβ17-35 β-hairpins, respectively. These hairpins are similar in composition but differ in hairpin alignment, altering the facial arrangements of the side chains of the residues that they contain. X-ray crystallography and SDS-PAGE demonstrate that the difference in facial arrangement between these peptides leads to distinct oligomer formation. In the crystal state, Aβm17-36 forms triangular trimers that further assemble to form hexamers, while Aβm17-35 forms tetrameric β-barrels. In SDS-PAGE, Aβm17-36 assembles to form a ladder of oligomers, while Aβm17-35 either assembles to form a dimer or does not assemble at all. The differences in the behavior of Aβm17-36 and Aβm17-35 suggest β-hairpin alignment as a source of the observed heterogeneity of Aβ oligomers.

Published
2024

2. Effects of N-Terminal Residues on the Assembly of Constrained β-Hairpin Peptides Derived from Aβ.

Author

Samdin, Tuan, Wierzbicki, Michal, Kreutzer, Adam, Howitz, William, Valenzuela, Mike, Smith, Alberto, Sahrai, Victoria, Truex, Nicholas, Klun, Matthew, and Nowick, James

Subjects
Amyloid beta-Peptides, Crystallography, X-Ray, Humans, Models, Molecular, Protein Conformation
Abstract

This paper describes the synthesis, solution-phase biophysical studies, and X-ray crystallographic structures of hexamers formed by macrocyclic β-hairpin peptides derived from the central and C-terminal regions of Aβ, which bear tails derived from the N-terminus of Aβ. Soluble oligomers of the β-amyloid peptide, Aβ, are thought to be the synaptotoxic species responsible for neurodegeneration in Alzheimers disease. Over the last 20 years, evidence has accumulated that implicates the N-terminus of Aβ as a region that may initiate the formation of damaging oligomeric species. We previously studied, in our laboratory, macrocyclic β-hairpin peptides derived from Aβ16-22 and Aβ30-36, capable of forming hexamers that can be observed by X-ray crystallography and SDS-PAGE. To better mimic oligomers of full length Aβ, we use an orthogonal protecting group strategy during the synthesis to append residues from Aβ1-14 to the parent macrocyclic β-hairpin peptide 1, which comprises Aβ16-22 and Aβ30-36. The N-terminally extended peptides N+1, N+2, N+4, N+6, N+8, N+10, N+12, and N+14 assemble to form dimers, trimers, and hexamers in solution-phase studies. X-ray crystallography reveals that peptide N+1 assembles to form a hexamer that is composed of dimers and trimers. These observations are consistent with a model in which the assembly of Aβ oligomers is driven by hydrogen bonding and hydrophobic packing of the residues from the central and C-terminal regions, with the N-terminus of Aβ accommodated by the oligomers as an unstructured tail.

Published
2020

3. Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons

Author

Truex, Nicholas L., primary, Mohapatra, Somesh, additional, Melo, Mariane, additional, Rodriguez, Jacob, additional, Li, Na, additional, Abraham, Wuhbet, additional, Sementa, Deborah, additional, Touti, Faycal, additional, Keskin, Derin B., additional, Wu, Catherine J., additional, Irvine, Darrell J., additional, Gómez-Bombarelli, Rafael, additional, and Pentelute, Bradley L., additional

Published
2024
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4. Transmembrane Proteins: Amyloids Hidden in Plain Sight?

Author

Truex, Nicholas L and Nowick, James S

Subjects
Biochemistry and Cell Biology, Biomedical and Clinical Sciences, Medicinal and Biomolecular Chemistry, Biological Sciences, Chemical Sciences, Medical Biochemistry and Metabolomics, Amyloid, Humans, Membrane Proteins, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Spectroscopy, Fourier Transform Infrared, X-Ray Diffraction, Biochemistry & Molecular Biology, Biochemistry and cell biology, Medical biochemistry and metabolomics, Medicinal and biomolecular chemistry
Published
2017

5. A Tetramer Derived from Islet Amyloid Polypeptide.

Author

Wang, Yilin, Kreutzer, Adam, Truex, Nicholas, and Nowick, James

Subjects
Crystallography, X-Ray, Humans, Hydrogen Bonding, Hydrophobic and Hydrophilic Interactions, Islet Amyloid Polypeptide, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation
Abstract

Aggregation of the islet amyloid polypeptide (IAPP) to form fibrils and oligomers is important in the progression of type 2 diabetes. This article describes X-ray crystallographic and solution-state NMR studies of peptides derived from residues 11-17 of IAPP that assemble to form tetramers. Incorporation of residues 11-17 of IAPP (RLANFLV) into a macrocyclic β-sheet peptide results in a monomeric peptide that does not self-assemble to form oligomers. Mutation of Arg11 to the uncharged isostere citrulline gives peptide homologues that assemble to form tetramers in both the crystal state and in aqueous solution. The tetramers consist of hydrogen-bonded dimers that sandwich together through hydrophobic interactions. The tetramers share several features with structures reported for IAPP fibrils and demonstrate the importance of hydrogen bonding and hydrophobic interactions in the oligomerization of IAPP-derived peptides.

Published
2017

6. Square channels formed by a peptide derived from transthyretin

Author

Yoo, Stan, Kreutzer, Adam G, Truex, Nicholas L, and Nowick, James S

Subjects
Cancer, Chemical Sciences
Abstract

High-resolution structures of peptide supramolecular assemblies are key to understanding amyloid diseases and designing peptide-based materials. This paper explores the supramolecular assembly of a macrocyclic β-sheet peptide derived from transthyretin (TTR). The peptide mimics the β-hairpin formed by the β-strands G and H of TTR, which form the interface of the TTR tetramer. X-ray crystallography reveals that the peptide does not form a tetramer, but rather assembles to form square channels. The square channels are formed by extended networks of β-sheets and pack in a "tilted windows" pattern. This unexpected structure represents an emergent property of the peptide and broadens the scope of known supramolecular assemblies of β-sheets.

Published
2016

8. Coassembly of Peptides Derived from β-Sheet Regions of β-Amyloid.

Author

Truex, Nicholas and Nowick, James

Subjects
Amino Acid Sequence, Amyloid beta-Peptides, Isomerism, Molecular Dynamics Simulation, Peptide Fragments, Protein Conformation, beta-Strand, Protein Multimerization
Abstract

In this paper, we investigate the coassembly of peptides derived from the central and C-terminal regions of the β-amyloid peptide (Aβ). In the preceding paper, J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b06000 , we established that peptides containing residues 17-23 (LVFFAED) from the central region of Aβ and residues 30-36 (AIIGLMV) from the C-terminal region of Aβ assemble to form homotetramers consisting of two hydrogen-bonded dimers. Here, we mix these tetramer-forming peptides and determine how they coassemble. Incorporation of a single 15N isotopic label into each peptide provides a spectroscopic probe with which to elucidate the coassembly of the peptides by 1H,15N HSQC. Jobs method of continuous variation and nonlinear least-squares fitting reveal that the peptides form a mixture of heterotetramers in 3:1, 2:2, and 1:3 stoichiometries, in addition to the homotetramers. These studies also establish the relative stability of each tetramer and show that the 2:2 heterotetramer predominates. 15N-Edited NOESY shows the 2:2 heterotetramer comprises two different homodimers, rather than two heterodimers. The peptides within the heterotetramer segregate in forming the homodimer subunits, but the two homodimers coassemble in forming the heterotetramer. These studies show that the central and C-terminal regions of Aβ can preferentially segregate within β-sheets and that the resulting segregated β-sheets can further coassemble.

Published
2016

11. Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons

Author

Truex, Nicholas L, primary, Mohapatra, Somesh, additional, Melo, Mariane, additional, Rodriguez, Jacob, additional, Li, Na, additional, Abraham, Wuhbet, additional, Sementa, Deborah, additional, Touti, Faycal, additional, Keskin, Derin B, additional, Wu, Catherine J, additional, Irvine, Darrell J, additional, Gomez-Bombarelli, Rafael, additional, and Pentelute, Bradley L., additional

Published
2023
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13. Assembly and Coassembly of Peptides Derived from β-Sheet Regions of β-Amyloid

Author

Truex, Nicholas L.

Subjects
Chemistry, Job's Method of Continuous Variation, NMR Spectroscopy, Nonlinear Least-Squares Fitting, Peptides, β-Amyloid, β-Sheets
Abstract

Interactions among β-sheets are critical in driving the aggregation of the β-amyloid peptide (Aβ) to form oligomers and fibrils in Alzheimer’s disease. Two main regions of this 40- or 42-residue peptide are known to adopt β-sheet structure and promote aggregation: the central region and the C-terminal region. The central region comprises the hydrophobic pentapeptide LVFFA (Aβ17–23), and the C-terminal region comprises the hydrophobic undecapeptide AIIGLMVGGVV (Aβ30–40) or the hydrophobic tridecapeptide AIIGLMVGGVVIA (Aβ30–42). This dissertation is devoted to studies of the assembly and coassembly of peptides derived from the central and C-terminal regions of Aβ, which are critical in the formation of oligomers and fibrils in Alzheimer’s disease pathology.Chapters 2 and 3 of this dissertation describe a two-part investigation of peptides derived from the central and C-terminal regions of Aβ. The first part, called “Assembly of Peptides Derived from β-Sheet Regions of β- Amyloid”, introduces two macrocyclic β-sheet peptides that contain residues 17–23 (LVFFAED) from the central region and residues 30–36 from the C-terminal region, and elucidates how each peptide self-assembles in aqueous solution. 1H NMR spectroscopy shows that the peptides assemble to form tetramers. Incorporation of a single isotopic label into each peptide and 15N-edited NMR spectroscopy facilitated the identification and quantification of the monomers and tetramers. Molecular modeling further elucidates the structures of the tetramers. These studies provide insights into the peptide interactions and supramolecular assembly of an important peptide in an important amyloid disease.The second part, called “Coassembly of Peptides Derived from β-Sheet Regions of β-Amyloid”, asks how the macrocyclic β-sheet peptides coassemble upon mixing. This question is important because the two regions generally segregate in most fibril structures of Aβ but coassemble in the oligomers. The two macrocyclic β-sheet peptides form a complex mixture homotetramers and heterotetramers upon mixing. 15N-Editted NMR spectroscopy shows that three heterotetramers form in addition to the homotetramers. Job’s method of continuous variation and nonlinear least-squares fitting to help establish the identity of each heterotetramer, and also reveal a surprising preference for one particular heterotetramer. These studies illustrate the role of molecular recognition in amyloid assembly and push the boundaries for experimental studies involving complex supramolecular equilibria.For the remainder of this dissertation, the focus of the research shifts to a study on macrocyclic β-sheets that contain N-methyl blocking groups, rather than Hao. These studies were intended to conclude ongoing efforts to characterize analogues of peptides derived from Aβ17–36. These analogues were studied by X-ray crystallography and 1H NMR spectroscopy, and also with biophysical and biological techniques. DOSY NMR studies revealed these peptides oligomerize in solution, which have not been observed previously by other biophysical techniques. Additional NMR experiments, and corroboratory analytical ultracentrifugation studies, are needed to further establish this finding. These studies show that evaluating the oligomers N-methyl peptides is possible by 1H NMR spectroscopy. These efforts to correlate solution-phase biophysical techniques with 1H NMR spectroscopy may prove useful to other members the Nowick lab.

Published
2017

14. IgG-Engineered Protective Antigen for Cytosolic Delivery of Proteins into Cancer Cells

Author

Massachusetts Institute of Technology. Department of Chemistry, Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Ragon Institute of MGH, MIT and Harvard, Massachusetts Institute of Technology. Department of Materials Science and Engineering, Massachusetts Institute of Technology. Department of Biological Engineering, Lu, Zeyu, Truex, Nicholas L, Melo, Mariane B, Cheng, Yiran, Li, Na, Irvine, Darrell J, Pentelute, Bradley L, Massachusetts Institute of Technology. Department of Chemistry, Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Ragon Institute of MGH, MIT and Harvard, Massachusetts Institute of Technology. Department of Materials Science and Engineering, Massachusetts Institute of Technology. Department of Biological Engineering, Lu, Zeyu, Truex, Nicholas L, Melo, Mariane B, Cheng, Yiran, Li, Na, Irvine, Darrell J, and Pentelute, Bradley L

Abstract

© 2020 American Chemical Society. All rights reserved. Therapeutic immunotoxins composed of antibodies and bacterial toxins provide potent activity against malignant cells, but joining them with a defined covalent bond while maintaining the desired function is challenging. Here, we develop novel immunotoxins by dovetailing full-length immunoglobulin G (IgG) antibodies and nontoxic anthrax proteins, in which the C terminus of the IgG heavy chain is connected to the side chain of anthrax toxin protective antigen. This strategy enabled efficient conjugation of protective antigen variants to trastuzumab (Tmab) and cetuximab (Cmab) antibodies. The conjugates effectively perform intracellular delivery of edema factor and N terminus of lethal factor (LFN) fused with diphtheria toxin and Ras/Rap1-specific endopeptidase. Each conjugate shows high specificity for cells expressing human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), respectively, and potent activity across six Tmab- and Cmab-resistant cell lines. The conjugates also exhibit increased pharmacokinetics and pronounced in vivo safety, which shows promise for further therapeutic development.

Published
2022

15. Targeting Cancer Gene Dependencies with Anthrax-Mediated Delivery of Peptide Nucleic Acids

Author

Lu, Zeyu, Paolella, Brenton R., Truex, Nicholas L., Loftis, Alexander R., Liao, Xiaoli, Rabideau, Amy E., Brown, Meredith S., Busanovich, John, Beroukhim, Rameen, Pentelute, Bradley L., Lu, Zeyu, Paolella, Brenton R., Truex, Nicholas L., Loftis, Alexander R., Liao, Xiaoli, Rabideau, Amy E., Brown, Meredith S., Busanovich, John, Beroukhim, Rameen, and Pentelute, Bradley L.

Abstract

Copyright © 2020 American Chemical Society. Antisense oligonucleotide therapies are important cancer treatments, which can suppress genes in cancer cells that are critical for cell survival. SF3B1 has recently emerged as a promising gene target that encodes a key splicing factor in the SF3B protein complex. Over 10% of cancers have lost one or more copies of the SF3B1 gene, rendering these cancers vulnerable after further suppression. SF3B1 is just one example of a CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) gene, but over 120 additional candidate CYCLOPS genes are known. Antisense oligonucleotide therapies for cancer offer the promise of effective suppression for CYCLOPS genes, but developing these treatments is difficult due to their limited permeability into cells and poor cytosolic stability. Here, we develop an effective approach to suppress CYCLOPS genes by delivering antisense peptide nucleic acids (PNAs) into the cytosol of cancer cells. We achieve efficient cytosolic PNA delivery with the two main nontoxic components of the anthrax toxin: protective antigen (PA) and the 263-residue N-terminal domain of lethal factor (LFN). Sortase-mediated ligation readily enables the conjugation of PNAs to the C terminus of the LFN protein. LFN and PA work together in concert to translocate PNAs into the cytosol of mammalian cells. Antisense SF3B1 PNAs delivered with the LFN/PA system suppress the SF3B1 gene and decrease cell viability, particularly of cancer cells with partial copy-number loss of SF3B1. Moreover, antisense SF3B1 PNAs delivered with a HER2-binding PA variant selectively target cancer cells that overexpress the HER2 cell receptor, demonstrating receptor-specific targeting of cancer cells. Taken together, our efforts illustrate how PA-mediated delivery of PNAs provides an effective and general approach for delivering antisense PNA therapeutics and for targeting gene dependencies in cancer.

Published
2022

16. Palladium–peptide oxidative addition complexes for bioconjugation

Author

Massachusetts Institute of Technology. Department of Chemistry, Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Rojas, Anthony J, Wolfe, Justin M, Dhanjee, Heemal H, Buslov, Ivan, Truex, Nicholas L, Liu, Richard Y, Massefski, Walter, Pentelute, Bradley L, Buchwald, Stephen L, Massachusetts Institute of Technology. Department of Chemistry, Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Rojas, Anthony J, Wolfe, Justin M, Dhanjee, Heemal H, Buslov, Ivan, Truex, Nicholas L, Liu, Richard Y, Massefski, Walter, Pentelute, Bradley L, and Buchwald, Stephen L

Abstract

Peptides bearing palladium oxidative addition complexes can be synthesized from the parent aryl halide containing substrates and react with thiol functional groups of small molecules, peptides, and proteins at low micromolar concentrations.

Published
2022

20. Targeting cancer gene dependencies with anthrax-mediated delivery of peptide nucleic acids

Author

Lu, Zeyu, Paolella, Brenton R, Truex, Nicholas L, Loftis, Alexander R, Liao, Xiaoli, Rabideau, Amy E, Brown, Meredith S, Busanovich, John, Beroukhim, Rameen, Pentelute, Bradley L, Lu, Zeyu, Paolella, Brenton R, Truex, Nicholas L, Loftis, Alexander R, Liao, Xiaoli, Rabideau, Amy E, Brown, Meredith S, Busanovich, John, Beroukhim, Rameen, and Pentelute, Bradley L

Abstract

Copyright © 2020 American Chemical Society. Antisense oligonucleotide therapies are important cancer treatments, which can suppress genes in cancer cells that are critical for cell survival. SF3B1 has recently emerged as a promising gene target that encodes a key splicing factor in the SF3B protein complex. Over 10% of cancers have lost one or more copies of the SF3B1 gene, rendering these cancers vulnerable after further suppression. SF3B1 is just one example of a CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) gene, but over 120 additional candidate CYCLOPS genes are known. Antisense oligonucleotide therapies for cancer offer the promise of effective suppression for CYCLOPS genes, but developing these treatments is difficult due to their limited permeability into cells and poor cytosolic stability. Here, we develop an effective approach to suppress CYCLOPS genes by delivering antisense peptide nucleic acids (PNAs) into the cytosol of cancer cells. We achieve efficient cytosolic PNA delivery with the two main nontoxic components of the anthrax toxin: protective antigen (PA) and the 263-residue N-terminal domain of lethal factor (LFN). Sortase-mediated ligation readily enables the conjugation of PNAs to the C terminus of the LFN protein. LFN and PA work together in concert to translocate PNAs into the cytosol of mammalian cells. Antisense SF3B1 PNAs delivered with the LFN/PA system suppress the SF3B1 gene and decrease cell viability, particularly of cancer cells with partial copy-number loss of SF3B1. Moreover, antisense SF3B1 PNAs delivered with a HER2-binding PA variant selectively target cancer cells that overexpress the HER2 cell receptor, demonstrating receptor-specific targeting of cancer cells. Taken together, our efforts illustrate how PA-mediated delivery of PNAs provides an effective and general approach for delivering antisense PNA therapeutics and for targeting gene dependencies in cancer.

Published
2021

21. IgG-Engineered Protective Antigen for Cytosolic Delivery of Proteins into Cancer Cells

Author

Lu, Zeyu, Truex, Nicholas L, Melo, Mariane B, Cheng, Yiran, Li, Na, Irvine, Darrell J, Pentelute, Bradley L, Lu, Zeyu, Truex, Nicholas L, Melo, Mariane B, Cheng, Yiran, Li, Na, Irvine, Darrell J, and Pentelute, Bradley L

Abstract

© 2020 American Chemical Society. All rights reserved. Therapeutic immunotoxins composed of antibodies and bacterial toxins provide potent activity against malignant cells, but joining them with a defined covalent bond while maintaining the desired function is challenging. Here, we develop novel immunotoxins by dovetailing full-length immunoglobulin G (IgG) antibodies and nontoxic anthrax proteins, in which the C terminus of the IgG heavy chain is connected to the side chain of anthrax toxin protective antigen. This strategy enabled efficient conjugation of protective antigen variants to trastuzumab (Tmab) and cetuximab (Cmab) antibodies. The conjugates effectively perform intracellular delivery of edema factor and N terminus of lethal factor (LFN) fused with diphtheria toxin and Ras/Rap1-specific endopeptidase. Each conjugate shows high specificity for cells expressing human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), respectively, and potent activity across six Tmab- and Cmab-resistant cell lines. The conjugates also exhibit increased pharmacokinetics and pronounced in vivo safety, which shows promise for further therapeutic development.

Published
2021

22. Anthrax Protective Antigen Retargeted with Single‐Chain Variable Fragments Delivers Enzymes to Pancreatic Cancer Cells

Author

Massachusetts Institute of Technology. Department of Chemistry, Massachusetts Institute of Technology. Department of Chemical Engineering, Loftis, Alexander Robert, Santos, Michael, Truex, Nicholas, Biancucci, Marco, Satchell, Karla J. F., Pentelute, Bradley L., Massachusetts Institute of Technology. Department of Chemistry, Massachusetts Institute of Technology. Department of Chemical Engineering, Loftis, Alexander Robert, Santos, Michael, Truex, Nicholas, Biancucci, Marco, Satchell, Karla J. F., and Pentelute, Bradley L.

Abstract

The nontoxic, anthrax protective antigen/lethal factor N-terminal domain (PA/LFN ) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LFN to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single-chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer-relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA-scFv fusions enable cell-specific delivery of diphtheria toxin catalytic domain and Ras/Rap1-specific endopeptidase to pancreatic cancer cells. Importantly, mPA-scFv fusion-based treatments display potent cell-specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells., NSF (Grant CHE-1351807)

Published
2020

23. Automated Flow Synthesis of Tumor Neoantigen Peptides for Personalized Immunotherapy

Author

Massachusetts Institute of Technology. Department of Chemistry, Koch Institute for Integrative Cancer Research at MIT, Truex, Nicholas, Holden, Rebecca Lynn, Wang, Bin-You, Chen, Pu-Guang, Hanna, Stephanie, Hu, Zhuting, Shetty, Keerthi, Olive, Oriol, Neuberg, Donna, Hacohen, Nir, Keskin, Derin B., Ott, Patrick A., Wu, Catherine J., Pentelute, Bradley L., Massachusetts Institute of Technology. Department of Chemistry, Koch Institute for Integrative Cancer Research at MIT, Truex, Nicholas, Holden, Rebecca Lynn, Wang, Bin-You, Chen, Pu-Guang, Hanna, Stephanie, Hu, Zhuting, Shetty, Keerthi, Olive, Oriol, Neuberg, Donna, Hacohen, Nir, Keskin, Derin B., Ott, Patrick A., Wu, Catherine J., and Pentelute, Bradley L.

Abstract

High-throughput genome sequencing and computation have enabled rapid identification of targets for personalized medicine, including cancer vaccines. Synthetic peptides are an established mode of cancer vaccine delivery, but generating the peptides for each patient in a rapid and affordable fashion remains difficult. High-throughput peptide synthesis technology is therefore urgently needed for patient-specific cancer vaccines to succeed in the clinic. Previously, we developed automated flow peptide synthesis technology that greatly accelerates the production of synthetic peptides. Herein, we show that this technology permits the synthesis of high-quality peptides for personalized medicine. Automated flow synthesis produces 30-mer peptides in less than 35 minutes and 15- to 16-mer peptides in less than 20 minutes. The purity of these peptides is comparable with or higher than the purity of peptides produced by other methods. This work illustrates how automated flow synthesis technology can enable customized peptide therapies by accelerating synthesis and increasing purity. We envision that implementing this technology in clinical settings will greatly increase capacity to generate clinical-grade peptides on demand, which is a key step in reaching the full potential of personalized vaccines for the treatment of cancer and other diseases., National Science Foundation (Grant 1122374), National Institutes of Health (Grants R21-CA216772-01A1 and NCI-1RO1CA155010-02), National Cancer Institute (Grants R21 CA216772-01A1 and SPORE-2P50CA101942-11A1), NHLBI (Grant 5R01HL103532-03)

Published
2020

28. A novel, safe, fast and efficient treatment for Her2‐positive and negative bladder cancer utilizing an EGF‐anthrax toxin chimera

Author

Jack, Sherwin, primary, Madhivanan, Kayalvizhi, additional, Ramadesikan, Swetha, additional, Subramanian, Sneha, additional, Edwards, Daniel F., additional, Elzey, Bennett D., additional, Dhawan, Deepika, additional, McCluskey, Andrew, additional, Kischuk, Erin M., additional, Loftis, Alexander R., additional, Truex, Nicholas, additional, Santos, Michael, additional, Lu, Mike, additional, Rabideau, Amy, additional, Pentelute, Bradley, additional, Collier, John, additional, Kaimakliotis, Hristos, additional, Koch, Michael, additional, Ratliff, Timothy L., additional, Knapp, Deborah W., additional, and Aguilar, Ruben C., additional

Published
2019
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29. A novel, safe, fast and efficient treatment for Her2‐positive and negative bladder cancer utilizing an EGF‐anthrax toxin chimera.

Author

Jack, Sherwin, Madhivanan, Kayalvizhi, Ramadesikan, Swetha, Subramanian, Sneha, Edwards, Daniel F., Elzey, Bennett D., Dhawan, Deepika, McCluskey, Andrew, Kischuk, Erin M., Loftis, Alexander R., Truex, Nicholas, Santos, Michael, Lu, Mike, Rabideau, Amy, Pentelute, Bradley, Collier, John, Kaimakliotis, Hristos, Koch, Michael, Ratliff, Timothy L., and Knapp, Deborah W.

Subjects
INTRAVESICAL administration, BLADDER cancer, EPIDERMAL growth factor, EPIDERMAL growth factor receptors, TOXINS, CANCER cells
Abstract

Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin‐shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF‐conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF‐based technologies and other toxin‐derivatives. In contrast to known agents, this EGF‐toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC50 < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor‐free mice and dogs with the agent resulted in no toxicity. In addition, the EGF‐toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF‐toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF–anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer. What's new? Bladder cancer is hard to treat and after surgery has a notoriously high recurrence rate (70%). Here the authors propose a new therapeutic strategy combining epidermal growth factor (EGF) with anthrax toxin. Since bladder cancer cells are exposed to urine and carry high levels of EGF receptor, the toxin after intravesical application was specifically taken up by cancer cells and induced rapid apoptosis, regardless of whether cancer cells expressed another EGF receptor, Her2, or not. This underscores the broad potential of the approach for treatment of bladder tumors in the future. [ABSTRACT FROM AUTHOR]

Published
2020
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34. Pd-catalyzed cross-coupling reactions exhibiting catalyst turnover numbers (TONs) exceeding one million.

Author

Shiqing Xu, Truex, Nicholas L., Mohan, Swathi, and Negishi, Ei-ichi

Subjects
*PALLADIUM catalysts, *COUPLING reactions (Chemistry), *ORGANOLITHIUM compounds, *ALKYNES, *ALKENYL group, *BROMIDES, *NITROBENZENE
Abstract

Pd-catalyzed cross-coupling giving aryl-aryl, alkenyl-aryl, aryl-alkenyl, alkynyl-aryl, and alkynyl-alkenyl products exhibited ultra-high turnover numbers (TONs) of 0.7×107~0.69×109 by using organozincs generated in situ by treatment of the corresponding organolithiums with dry ZnBr2. Additionally, the hydrozirconation-Pd-catalyzed cross-coupling tandem processes via treatment of 1-alkynes with iBu2AlH-ZrCp2Cl2 followed by selective (≥98%) alkenyl-alkenyl coupling with either (E)- or (Z)-ethyl 3-bromoacrylate exhibited high TONs of 0.9×105~0.81×107. Furthermore, Pd-catalyzed cross-coupling of 2-thienylzinc bromide and 1- iodo-4-nitrobenzene also showed a high TON of 0.87×105. [ABSTRACT FROM AUTHOR]

Published
2012

35. Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons.

Author

Truex NL, Mohapatra S, Melo M, Rodriguez J, Li N, Abraham W, Sementa D, Touti F, Keskin DB, Wu CJ, Irvine DJ, Gómez-Bombarelli R, and Pentelute BL

Abstract

Antigen processing is critical for producing epitope peptides that are presented by HLA molecules for T cell recognition. Therapeutic vaccines aim to harness these epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often reduces vaccine efficacy through limiting the quantity of epitopes released. Here, we set out to improve antigen processing by harnessing protein degradation signals called degrons from the ubiquitin-proteasome system. We used machine learning to generate a computational model that ascribes a proteasomal degradation score between 0 and 100. Epitope peptides with varying degron activities were synthesized and translocated into cells using nontoxic anthrax proteins: protective antigen (PA) and the N-terminus of lethal factor (LF N ). Immunogenicity studies revealed epitope sequences with a low score (<25) show pronounced T-cell activation but epitope sequences with a higher score (>75) provide limited activation. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity, through conserving the epitope region but varying the flanking sequence. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by vaccine therapeutics in clinical settings., Competing Interests: Competing interests B.L.P. is a co-founder and/or member of the scientific advisory board of several companies focusing on the development of protein and peptide therapeutics. DBK is a scientific advisor for Immunitrack and Breakbio. DBK owns equity in Affimed N.V., Agenus, Armata Pharmaceuticals, Breakbio, BioMarin Pharmaceutical, Celldex Therapeutics, Editas Medicine, Gilead Sciences, Immunitybio, ImmunoGen, IMV, Lexicon Pharmaceuticals, Neoleukin Therapeutics. BeiGene, a Chinese biotech company, supported unrelated SARS COV-2 research at TIGL.

Published
2023
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