ABSTRACT
The mechanism(s) by which certain small peptides and peptide mimics carry large cargoes across membranes through exclusively non-covalent interactions has been difficult to resolve. Here, we use the droplet-interface bilayer as a platform to characterize distinct mechanistic differences between two such carriers: Pep-1 and a guanidinium-rich peptide mimic we call D9. While both Pep-1 and D9 can carry an enzyme, horseradish peroxidase (HRP) across a lipid bilayer, we found that they do so by different mechanisms. Specifically, Pep-1 requires voltage or membrane asymmetry while D9 does not. In addition, D9 can facilitate HRP transport without pre-forming a complex with HRP. By contrast, complex formation is required by Pep-1. Both carriers are capable of forming pores in membranes but our data hints that these pores are not responsible for cargo transport. Overall, D9 appears to be a more potent and versatile transporter when compared with Pep-1 because D9 does not require an applied voltage or other forces to drive transport. Thus, D9 might be used to deliver cargo across membranes under conditions where Pep-1 would be ineffective.
Subject(s)
Cell Membrane/metabolism , Cysteamine/analogs & derivatives , Lipid Bilayers/metabolism , Peptides/metabolism , Amino Acid Sequence , Carrier Proteins/chemistry , Cell Membrane/chemistry , Cysteamine/chemistry , Cysteamine/metabolism , Guanidine/chemistry , Horseradish Peroxidase/metabolism , Lipid Bilayers/chemistry , Membrane Potentials , Models, Biological , Molecular Sequence Data , Molecular Structure , Peptides/chemistry , Protein TransportABSTRACT
Inhibition of the S-adenosyl methionine (SAM)-producing metabolic enzyme, methionine adenosyltransferase 2A (MAT2A), has received significant interest in the field of medicinal chemistry due to its implication as a synthetic lethal target in cancers with the deletion of the methylthioadenosine phosphorylase (MTAP) gene. Here, we report the identification of novel MAT2A inhibitors with distinct in vivo properties that may enhance their utility in treating patients. Following a high-throughput screening, we successfully applied the structure-based design lessons from our first-in-class MAT2A inhibitor, AG-270, to rapidly redesign and optimize our initial hit into two new lead compounds: a brain-penetrant compound, AGI-41998, and a potent, but limited brain-penetrant compound, AGI-43192. We hope that the identification and first disclosure of brain-penetrant MAT2A inhibitors will create new opportunities to explore the potential therapeutic effects of SAM modulation in the central nervous system (CNS).
Subject(s)
Methionine Adenosyltransferase , Neoplasms , Brain/metabolism , Drug Design , Humans , Neoplasms/drug therapy , S-Adenosylmethionine/metabolismABSTRACT
We introduce a new method for monitoring and quantitating the transport of materials across a model cell membrane. As a proof-of-concept, the cell-penetrating peptide, Pep-1, was used to carry horseradish peroxidase (HRP) across droplet-interface bilayers (DIBs). Two submicroliter, lipid-encased aqueous droplets form a membrane at the contacting interface, through which enzyme-peptide complexes pass during transport. Following transport, the droplets are separated and the captured enzymes are assayed by a fluorogenic reaction. The DIB method recapitulates the findings of earlier studies involving Pep-1, including the dependence of protein transport on voltage and membrane charge, while also contributing new insights. Specifically, we found that leaflet charge symmetry may play a role in Pep-1-mediated protein translocation. We anticipate that the DIB method may be useful for a variety of transport-based studies.
Subject(s)
Cysteamine/analogs & derivatives , Lipid Bilayers/metabolism , Peptides/metabolism , Biochemistry/methods , Cysteamine/metabolism , Horseradish Peroxidase/administration & dosage , Protein TransportABSTRACT
The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene and is co-deleted with CDKN2A in approximately 15% of all cancers. This co-deletion leads to aggressive tumors with poor prognosis that lack effective, molecularly targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We report the characterization of potent MAT2A inhibitors that substantially reduce levels of S-adenosylmethionine (SAM) and demonstrate antiproliferative activity in MTAP-deleted cancer cells and tumors. Using RNA sequencing and proteomics, we demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that DNA damage and mitotic defects ensue upon MAT2A inhibition in HCT116 MTAP-/- cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.
Subject(s)
DNA Damage/drug effects , Enzyme Inhibitors/pharmacology , Methionine Adenosyltransferase/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/genetics , Purine-Nucleoside Phosphorylase/genetics , RNA Splicing/drug effects , RNA, Messenger/genetics , Animals , Cell Line , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p16 , DNA Damage/genetics , Gene Deletion , HCT116 Cells , HEK293 Cells , Humans , Methionine Adenosyltransferase/genetics , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Neoplasms/drug therapy , Neoplasms/genetics , RNA Splicing/genetics , S-Adenosylmethionine/metabolismABSTRACT
Maxwell electromagnetism, describing the wave properties of light, was formulated 150 years ago. More than 60 years ago it was shown that interfaces between optical media (including dielectrics, metals, negative-index materials) can support surface electromagnetic waves, which now play crucial roles in plasmonics, metamaterials, and nano-photonics. Here we show that surface Maxwell waves at interfaces between homogeneous isotropic media described by real permittivities and permeabilities have a topological origin explained by the bulk-boundary correspondence. Importantly, the topological classification is determined by the helicity operator, which is generically non-Hermitian even in lossless optical media. The corresponding topological invariant, which determines the number of surface modes, is a [Formula: see text] number (or a pair of [Formula: see text] numbers) describing the winding of the complex helicity spectrum across the interface. Our theory provides a new twist and insights for several areas of wave physics: Maxwell electromagnetism, topological quantum states, non-Hermitian wave physics, and metamaterials.
ABSTRACT
We have designed two novel devices which extend the applications for the droplet-interface bilayer (DIB) as a model membrane system. The add-chip allows successive reagent additions to one side of the lipid bilayer during an experiment while maintaining a simple setup with much lower volumes than in planar bilayer systems. The flow-chip is capable of multiple complete solution perfusions concurrently with electrophysiology measurements. Both devices preserve all of the key advantages that DIBs have relative to planar membranes, including low volume, leaflet asymmetry and the ability to separate the monolayers prior to further analysis of a droplet's contents. As a demonstration, we use these devices to monitor and quantitate molecular transport across DIBs.