ABSTRACT
Ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, provides a potential treatment avenue for drug-resistant cancers and may play a role in the pathology of some degenerative diseases. Identifying the subcellular membranes essential for ferroptosis and the sequence of their peroxidation will illuminate drug discovery strategies and ferroptosis-relevant disease mechanisms. In this study, we employed fluorescence and stimulated Raman scattering imaging to examine the structure-activity-distribution relationship of ferroptosis-modulating compounds. We found that, although lipid peroxidation in various subcellular membranes can induce ferroptosis, the endoplasmic reticulum (ER) membrane is a key site of lipid peroxidation. Our results suggest an ordered progression model of membrane peroxidation during ferroptosis that accumulates initially in the ER membrane and later in the plasma membrane. Thus, the design of ER-targeted inhibitors and inducers of ferroptosis may be used to optimally control the dynamics of lipid peroxidation in cells undergoing ferroptosis.
Subject(s)
Ferroptosis , Lipid Peroxidation/physiology , Cell Death , Cell Membrane/metabolism , Iron/metabolismABSTRACT
The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a 'colour barrier', owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.
Subject(s)
Molecular Imaging/methods , Spectrum Analysis, Raman/methods , Vibration , Animals , Brain/cytology , Cell Line , Cell Survival , Coculture Techniques , Color , Coloring Agents/analysis , Coloring Agents/chemistry , DNA/metabolism , Electrons , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Humans , Infrared Rays , Mice , Neurons/cytology , Organ Specificity , Proteins/metabolismABSTRACT
Semiconductors demonstrate great potentials as chemical mechanism-based surface-enhanced Raman scattering (SERS) substrates in determination of biological species in complex living systems with high selectivity. However, low sensitivity is the bottleneck for their practical applications, compared with that of noble metal-based Raman enhancement ascribed to electromagnetic mechanism. Herein, a novel Cu2 O nanoarray with free carrier density of 1.78×1021 â cm-3 comparable to that of noble metals was self-assembled, creating a record in enhancement factor (EF) of 3.19×1010 among semiconductor substrates. The significant EF was mainly attributed to plasmon-induced hot electron transfer (PIHET) in semiconductor which was never reported before. This Cu2 O nanoarray was subsequently developed as a highly sensitive and selective SERS chip for non-enzyme and amplification-free SARS-CoV-2 RNA quantification with a detection limit down to 60â copies/mL within 5â min. This unique Cu2 O nanoarray demonstrated the significant Raman enhancement through PIHET process, enabling rapid and sensitive point-of-care testing of emerging virus variants.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , COVID-19/diagnosis , RNA, Viral , Spectrum Analysis, Raman , SemiconductorsABSTRACT
All molecules consist of chemical bonds, and much can be learned from mapping the spatiotemporal dynamics of these bonds. Since its invention a decade ago, stimulated Raman scattering (SRS) microscopy has become a powerful modality for imaging chemical bonds with high sensitivity, resolution, speed and specificity. We introduce the fundamentals of SRS microscopy and review innovations in SRS microscopes and imaging probes. We highlight examples of exciting biological applications, and share our vision for potential future breakthroughs for this technology.
Subject(s)
Macromolecular Substances/analysis , Molecular Imaging/methods , Nonlinear Optical Microscopy/methods , Animals , HumansABSTRACT
Optical multiplexing has a large impact in photonics, the life sciences and biomedicine. However, current technology is limited by a 'multiplexing ceiling' from existing optical materials. Here we engineered a class of polyyne-based materials for optical supermultiplexing. We achieved 20 distinct Raman frequencies, as 'Carbon rainbow', through rational engineering of conjugation length, bond-selective isotope doping and end-capping substitution of polyynes. With further probe functionalization, we demonstrated ten-color organelle imaging in individual living cells with high specificity, sensitivity and photostability. Moreover, we realized optical data storage and identification by combinatorial barcoding, yielding to our knowledge the largest number of distinct spectral barcodes to date. Therefore, these polyynes hold great promise in live-cell imaging and sorting as well as in high-throughput diagnostics and screening.
Subject(s)
Fluorescent Dyes/chemistry , Optical Imaging/instrumentation , Optical Imaging/methods , Polyynes/chemistry , Spectrum Analysis, Raman/methods , Cell Survival , HeLa Cells , Humans , Optics and PhotonicsABSTRACT
Optical microscopy has served biomedical research for decades due to its high temporal and spatial resolutions. Among various optical imaging techniques, fluorescence imaging offers superb sensitivity down to single molecule level but its multiplexing capacity is limited by intrinsically broad bandwidth. To simultaneously capture a vast number of targets, the newly emerging vibrational microscopy technique draws increasing attention as vibration spectroscopy features narrow transition linewidth. Nonetheless, unlike fluorophores that have been studied for centuries, a systematic investigation on vibrational probes is underemphasized. Herein, we reviewed some of the recent developments of vibrational probes for multiplex imaging applications, particularly those serving stimulated Raman scattering (SRS) microscopy, which is one of the most promising vibrational imaging techniques. We wish to summarize the general guidelines for developing bioorthogonal vibrational probes with high sensitivity, chemical specificity and most importantly, tunability to fulfill super-multiplexed optical imaging. Future directions to significantly improve the performance are also discussed.
Subject(s)
Indicators and Reagents/chemistry , Microscopy/methods , Vibration , Alkynes/chemistry , Animals , Biosensing Techniques/methods , Cell Line , Humans , Nanotechnology/methods , Sensitivity and Specificity , Single Molecule Imaging/methods , Spectrum Analysis, Raman/methodsABSTRACT
Stimulated Raman Scattering (SRS) coupled with alkyne tags has been an emerging imaging technique to visualize small-molecule species with high sensitivity and specificity. Here we describe the development of a ratiometric Raman probe for visualizing hydrogen sulfide (H2S) species in living cells as the first alkyne-based sensor for SRS microscopy. This probe uses an azide unit as a selective reactive site, and it targets mitochondria with high specificity. The SRS ratiometric images show a strong response to H2S level changes in living cells.
Subject(s)
Alkynes/chemistry , Azides/chemistry , Hydrogen Sulfide/analysis , Luminescent Agents/chemistry , Mitochondria/metabolism , Spectrum Analysis, Raman/methods , Alkynes/chemical synthesis , Alkynes/toxicity , Animals , Azides/chemical synthesis , Azides/toxicity , COS Cells , Chlorocebus aethiops , HeLa Cells , Humans , Hydrogen Sulfide/chemistry , Hydrogen Sulfide/metabolism , Limit of Detection , Luminescent Agents/chemical synthesis , Luminescent Agents/toxicityABSTRACT
Sensitive and specific visualization of small biomolecules in living systems is highly challenging. We report stimulated Raman-scattering imaging of alkyne tags as a general strategy for studying a broad spectrum of small biomolecules in live cells and animals. We demonstrate this technique by tracking alkyne-bearing drugs in mouse tissues and visualizing de novo synthesis of DNA, RNA, proteins, phospholipids and triglycerides through metabolic incorporation of alkyne-tagged small precursors.
Subject(s)
Spectrum Analysis, Raman/methods , Alkynes , Animals , DNA/biosynthesis , Gene Expression Regulation/physiology , HeLa Cells , Humans , Mice , Molecular Structure , Naphthalenes , Proteins/metabolism , RNA/biosynthesis , TerbinafineABSTRACT
Innovations in light microscopy have tremendously revolutionized the way researchers study biological systems with subcellular resolution. In particular, fluorescence microscopy with the expanding choices of fluorescent probes has provided a comprehensive toolkit to tag and visualize various molecules of interest with exquisite specificity and high sensitivity. Although fluorescence microscopy is currently the method of choice for cellular imaging, it faces fundamental limitations for studying the vast number of small biomolecules. This is because common fluorescent labels, which are relatively bulky, could introduce considerable perturbation to or even completely alter the native functions of vital small biomolecules. Hence, despite their immense functional importance, these small biomolecules remain largely undetectable by fluorescence microscopy. To address this challenge, a bioorthogonal chemical imaging platform has recently been introduced. By coupling stimulated Raman scattering (SRS) microscopy, an emerging nonlinear Raman microscopy technique, with tiny and Raman-active vibrational probes (e.g., alkynes and stable isotopes), bioorthogonal chemical imaging exhibits superb sensitivity, specificity, and biocompatibility for imaging small biomolecules in live systems. In this Account, we review recent technical achievements for visualizing a broad spectrum of small biomolecules, including ribonucleosides and deoxyribonucleosides, amino acids, fatty acids, choline, glucose, cholesterol, and small-molecule drugs in live biological systems ranging from individual cells to animal tissues and model organisms. Importantly, this platform is compatible with live-cell biology, thus allowing real-time imaging of small-molecule dynamics. Moreover, we discuss further chemical and spectroscopic strategies for multicolor bioorthogonal chemical imaging, a valuable technique in the era of "omics". As a unique tool for biological discovery, this platform has been applied to studying various metabolic processes under both physiological and pathological states, including protein synthesis activity of neuronal systems, protein aggregations in Huntington disease models, glucose uptake in tumor xenografts, and drug penetration through skin tissues. We envision that the coupling of SRS microscopy with vibrational probes would do for small biomolecules what fluorescence microscopy of fluorophores has done for larger molecular species.
Subject(s)
Molecular Probes/metabolism , Nonlinear Optical Microscopy/methods , Alkynes/chemistry , Amino Acids/metabolism , Animals , Carbon Radioisotopes , Deuterium , Fatty Acids/metabolism , HeLa Cells , Humans , Molecular Probes/chemistry , Nucleosides/metabolism , Proteins/metabolism , VibrationABSTRACT
As a superb tool to visualize and study the spatial-temporal distribution of chemicals, Raman microscopy has made a big impact in many disciplines of science. While label-free imaging has been the prevailing strategy in Raman microscopy, recent development and applications of vibrational/Raman tags, particularly when coupled with stimulated Raman scattering (SRS) microscopy, have generated intense excitement in biomedical imaging. SRS imaging of vibrational tags has enabled researchers to study a wide range of small biomolecules with high specificity, sensitivity and multiplex capability, at a single live cell level, tissue level or even in vivo. As reviewed in this article, this platform has facilitated imaging distribution and dynamics of small molecules such as glucose, lipids, amino acids, nucleic acids, and drugs that are otherwise difficult to monitor with other means. As both the vibrational tags and Raman instrumental development progress rapidly and synergistically, we anticipate that this technique will shed light onto an even broader spectrum of biomedical problems.
ABSTRACT
The "deltic guanidinium" ion is described here as a "macrostere" of the guanidinium ion. The use of the 2,4-dimethoxybenzyl protecting group allows for the synthesis of the fully unsubstituted parent compound and a variety of derivatives bearing multiple N-H functions for the first time. Deltic urea, deltic thiourea, and deltic benzamidine are also synthesized. A comparison of the physical properties of guanidinium and deltic guanidinium ions is provided. The use of a deltic guanidinium dendrimer for cell transport is demonstrated.
ABSTRACT
Glucose is a ubiquitous energy source for most living organisms. Its uptake activity closely reflects cellular metabolic demand in various physiopathological conditions. Extensive efforts have been made to specifically image glucose uptake, such as with positron emission tomography, magnetic resonance imaging, and fluorescence microscopy, but all have limitations. A new platform to visualize glucose uptake activity in live cells and tissues is presented that involves performing stimulated Raman scattering on a novel glucose analogue labeled with a small alkyne moiety. Cancer cells with differing metabolic activities can be distinguished. Heterogeneous uptake patterns are observed with clear cell-cell variations in tumor xenograft tissues, neuronal culture, and mouse brain tissues. By offering the distinct advantage of optical resolution but without the undesirable influence of fluorophores, this method will facilitate the study of energy demands of living systems with subcellular resolution.
Subject(s)
Glucose/metabolism , Glucose/pharmacokinetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Vibration , Animals , Cell Survival , Glucose/analogs & derivatives , HeLa Cells , Humans , Mice , Spectrum Analysis, Raman , Tissue DistributionABSTRACT
Choline is a small molecule that occupies a key position in the biochemistry of all living organisms. Recent studies have strongly implicated choline metabolites in cancer, atherosclerosis and nervous system development. To detect choline and its metabolites, existing physical methods such as magnetic resonance spectroscopy and positron emission tomography are often limited by the poor spatial resolution and substantial radiation dose. Fluorescence imaging, although with submicrometer resolution, requires introduction of bulky fluorophores and thus is difficult in labeling the small choline molecule. By combining the emerging bond-selective stimulated Raman scattering microscopy with metabolic incorporation of deuterated choline, herein we have achieved high resolution imaging of choline-containing metabolites in living mammalian cell lines, primary hippocampal neurons and the multicellular organism C. elegans. Different subcellular distributions of choline metabolites are observed between cancer cells and non-cancer cells, which may reveal a functional difference in the choline metabolism and lipid-mediated signaling events. In neurons, choline incorporation is visualized within both soma and neurites, where choline metabolites are more evenly distributed compared to proteins. Furthermore, choline localization is also observed in the pharynx region of C. elegans larvae, consistent with its organogenesis mechanism. These applications demonstrate the potential of isotope-based stimulated Raman scattering microscopy for future choline-related disease detection and development monitoring in vivo.
Subject(s)
Isotope Labeling , Metabolism , Spectrum Analysis, Raman/methods , Vibration , Animals , Cell Line , HumansABSTRACT
Protein degradation is a regulatory process essential to cell viability and its dysfunction is implicated in many diseases, such as aging and neurodegeneration. In this report, stimulated Raman scattering microscopy coupled with metabolic labeling with (13)C-phenylalanine is used to visualize protein degradation in living cells with subcellular resolution. We choose the ring breathing modes of endogenous (12)C-phenylalanine and incorporated (13)C-phenylalanine as protein markers for the original and nascent proteomes, respectively, and the decay of the former wasquantified through (12)C/((12)C+(13)C) ratio maps. We demonstrate time-dependent imaging of proteomic degradation in mammalian cells under steady-state conditions and various perturbations, including oxidative stress, cell differentiation, and huntingtin protein aggregation.
Subject(s)
Proteins/metabolism , Proteome/analysis , Spectrum Analysis, Raman , Carbon Isotopes/chemistry , HEK293 Cells , HeLa Cells , Humans , Hydrogen Peroxide/toxicity , Phenylalanine/chemistry , Proteins/analysis , Proteolysis/drug effectsABSTRACT
Optical imaging with photo-controllable probes has greatly advanced biological research. With superb chemical specificity of vibrational spectroscopy, stimulated Raman scattering (SRS) microscopy is particularly promising for super-multiplexed optical imaging with rich chemical information. Functional SRS imaging in response to light has been recently demonstrated, but multiplexed SRS imaging with reversible photocontrol remains unaccomplished. Here, we create a multiplexing palette of photoswitchable polyynes with 16 Raman frequencies by coupling asymmetric diarylethene with super-multiplexed Carbow (Carbow-switch). Through optimization of both electronic and vibrational spectroscopy, Carbow-switch displays excellent photoswitching properties under visible light control and SRS response with large frequency change and signal enhancement. Reversible and spatial-selective multiplexed SRS imaging of different organelles are demonstrated in living cells. We further achieve photo-selective time-lapse imaging of organelle dynamics during oxidative stress and protein phase separation. The development of Carbow-switch for photoswitchable SRS microscopy will open up new avenues to study complex interactions and dynamics in living cells with high spatiotemporal precision and multiplexing capability.
ABSTRACT
We review the current state of membrane protein structure determination using solid-state nuclear magnetic resonance (NMR) spectroscopy. Multidimensional magic-angle-spinning correlation NMR combined with oriented-sample experiments has made it possible to measure a full panel of structural constraints of membrane proteins directly in lipid bilayers. These constraints include torsion angles, interatomic distances, oligomeric structure, protein dynamics, ligand structure and dynamics, and protein orientation and depth of insertion in the lipid bilayer. Using solid-state NMR, researchers have studied potassium channels, proton channels, Ca(2+) pumps, G protein-coupled receptors, bacterial outer membrane proteins, and viral fusion proteins to elucidate their mechanisms of action. Many of these membrane proteins have also been investigated in detergent micelles using solution NMR. Comparison of the solid-state and solution NMR structures provides important insights into the effects of the solubilizing environment on membrane protein structure and dynamics.
Subject(s)
Membrane Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Animals , Humans , Lipid Bilayers/chemistry , Molecular Dynamics Simulation , Protein Conformation , Viral Fusion Proteins/chemistry , Viruses/chemistryABSTRACT
Membrane proteins change their conformations to respond to environmental cues, thus conformational plasticity is important for function. The influenza A M2 protein forms an acid-activated proton channel important for the virus lifecycle. Here we have used solid-state NMR spectroscopy to examine the conformational plasticity of membrane-bound transmembrane domain of M2 (M2TM). (13)C and (15)N chemical shifts indicate coupled conformational changes of several pore-facing residues due to changes in bilayer thickness, drug binding, and pH. The structural changes are attributed to the formation of a well-defined helical kink at G34 in the drug-bound state and in thick lipid bilayers, nonideal backbone conformation of the secondary-gate residue V27 in the presence of drug, and nonideal conformation of the proton-sensing residue H37 at high pH. The chemical shifts constrained the (Ï, ψ) torsion angles for three "basis" states, the equilibrium among which explains the multiple resonances per site in the NMR spectra under different combinations of bilayer thickness, drug binding, and pH conditions. Thus, conformational plasticity is important for the proton conduction and inhibition of M2TM. The study illustrates the utility of NMR chemical shifts for probing the structural plasticity and folding of membrane proteins.
Subject(s)
Chemistry, Pharmaceutical/methods , Influenza A virus/metabolism , Membranes, Artificial , Viral Matrix Proteins/metabolism , Amantadine/pharmacology , Antiviral Agents/pharmacology , Detergents/chemistry , Humans , Hydrogen-Ion Concentration , Lipid Bilayers/chemistry , Lipids/chemistry , Magnetic Resonance Spectroscopy/methods , Protein Binding , Protein Conformation , Protein Structure, TertiaryABSTRACT
The acid-activated proton channel formed by the influenza M2 protein is important for the life cycle of the virus. A single histidine, His37, in the M2 transmembrane domain (M2TM) is responsible for pH activation and proton selectivity of the channel. Recent studies suggested three models for how His37 mediates proton transport: a shuttle mechanism involving His37 protonation and deprotonation, a H-bonded imidazole-imidazolium dimer model, and a transporter model involving large protein conformational changes in synchrony with proton conduction. Using magic-angle-spinning (MAS) solid-state NMR spectroscopy, we examined the proton exchange and backbone conformational dynamics of M2TM in a virus-envelope-mimetic membrane. At physiological temperature and pH, (15)N NMR spectra show fast exchange of the imidazole (15)N between protonated and unprotonated states. To quantify the proton exchange rates, we measured the (15)N T(2) relaxation times and simulated them for chemical-shift exchange and fluctuating N-H dipolar fields under (1)H decoupling and MAS. The exchange rate is 4.5 × 10(5) s(-1) for Nδ1 and 1.0 × 10(5) s(-1) for Nε2, which are approximately synchronized with the recently reported imidazole reorientation. Binding of the antiviral drug amantadine suppressed both proton exchange and ring motion, thus interfering with the proton transfer mechanism. By measuring the relative concentrations of neutral and cationic His as a function of pH, we determined the four pK(a) values of the His37 tetrad in the viral membrane. Fitting the proton current curve using the charge-state populations from these pK(a)'s, we obtained the relative conductance of the five charge states, which showed that the +3 channel has the highest time-averaged unitary conductance. At physiologically relevant pH, 2D correlation spectra indicated that the neutral and cationic histidines do not have close contacts, ruling out the H-bonded dimer model. Moreover, a narrowly distributed nonideal helical structure coexists with a broadly distributed ideal helical conformation without interchange on the sub-10 ms time scale, thus excluding the transporter model in the viral membrane. These data support the shuttle mechanism of proton conduction, whose essential steps involve His-water proton exchange facilitated by imidazole ring reorientations.
Subject(s)
Histidine/chemistry , Nuclear Magnetic Resonance, Biomolecular , Protons , Viral Matrix Proteins/chemistry , Water/chemistry , Electric Conductivity , Hydrogen-Ion ConcentrationABSTRACT
Paramagnetic Cu(II) ions enhance nuclear spin relaxation in a distance-dependent fashion and can be used as a structural probe of proteins. Cu(II) can also serve as a functionally important ligand in proteins. Here we investigate the structural basis of Cu(II) inhibition of the influenza M2 proton channel through Cu(II)-induced paramagnetic relaxation enhancement (PRE). (13)C T(1) relaxation rates of the central residues of the transmembrane (TM) domain of M2 are significantly enhanced by Cu(II), and pronounced spectral broadening is observed for the proton-selective residue, His37. These data yielded quantitative distances of (13)C spins to the Cu(II) center and identified the Cu(II) binding site to be Nε2 of His37. This binding site is surrounded by four imidazole rings from the top and four indole rings of Trp41 from the bottom, thus explaining the high affinity of Cu(II) binding. Bound at this location, Cu(II) can inhibit proton currents by perturbing histidine-water proton exchange, preventing histidine conformational dynamics, and interfering with His-Trp cation-π interaction. The Cu(II) binding site is distinct from the binding site of the hydrophobic drug amantadine, which is about 10 Å N-terminal to His37. Consistently, Cu(II) and amantadine induce distinct conformational changes at several key residues, suggesting the possibility of designing new drugs that target the His37 site to inhibit amantadine-resistant mutant M2 proteins. In addition to the high-affinity His37 binding site, we also examined the weaker and nonspecific binding of Cu(II) to membrane-surface lipid phosphates and the extent of the resulting PRE to surface-proximal protein residues. This study demonstrates the feasibility of NMR studies of paramagnetic-ion-complexed membrane proteins, where the ion serves as both a functional ligand and a distance probe.
Subject(s)
Copper/chemistry , Influenza A virus/chemistry , Proton Pumps/chemistry , Proton Pumps/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Binding Sites , Copper/metabolism , Humans , Influenza A virus/metabolism , Influenza, Human/virology , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Protein Structure, Tertiary , Proton Pump Inhibitors , Viral Proteins/antagonists & inhibitorsABSTRACT
Influenza A virus M2 (A/M2) forms a homotetrameric proton selective channel in the viral membrane. It has been the drug target of antiviral drugs such as amantadine and rimantadine. However, most of the current virulent influenza A viruses carry drug-resistant mutations alongside the drug binding site, such as S31N, V27A, and L26F, etc., each of which might be dominant in a given flu season. Among these mutations, the V27A mutation was prevalent among transmissible viruses under drug selection pressure. Until now, V27A has not been successfully targeted by small molecule inhibitors, despite years of extensive medicinal chemistry research efforts and high throughput screening. Guided by molecular dynamics (MD) simulation of drug binding and the influence of drug binding on the dynamics of A/M2 from earlier experimental studies, we designed a series of potent spirane amine inhibitors targeting not only WT, but also both A/M2-27A and L26F mutants with IC(50)s similar to that seen for amantadine's inhibition of the WT channel. The potencies of these inhibitors were further demonstrated in experimental binding and plaque reduction assays. These results demonstrate the power of MD simulations to probe the mechanism of drug binding as well as the ability to guide design of inhibitors of targets that had previously appeared to be undruggable.