Searching for Blockers of Dengue and West Nile Virus Viroporins.

Flavivirus infections, such as those caused by dengue and West Nile viruses, emerge as new challenges for the global healthcare sector. It has been found that these two viruses encode ion channels collectively termed viroporins. Therefore, drug molecules that block such ion-channel activity can serve as potential antiviral agents and may play a primary role in therapeutic purposes. We screened 2839 FDA-approved drugs and compounds in advanced experimental phases using three bacteria-based channel assays to identify such ion channel blockers. We primarily followed a negative genetic screen in which the channel is harmful to the bacteria due to excessive membrane permeabilization that can be relieved by a blocker. Subsequently, we cross-checked the outcome with a positive genetic screen and a pH-dependent assay. The following drugs exhibited potential blocker activities: plerixafor, streptomycin, tranexamic acid, CI-1040, glecaprevir, kasugamycin, and mesna were effective against dengue virus DP1. In contrast, idasanutlin, benzbromarone, 5-azacytidine, and plerixafor were effective against West Nile Virus MgM. These drugs can serve as future antiviral therapeutic agents following subsequent in vitro and in vivo efficacy studies.

Blockers of the SARS-CoV-2 3a Channel Identified by Targeted Drug Repurposing.

The etiological agent of the COVID-19 pandemic is SARS-CoV-2. As a member of the Coronaviridae, the enveloped pathogen has several membrane proteins, of which two, E and 3a, were suggested to function as ion channels. In an effort to increase our treatment options, alongside providing new research tools, we have sought to inhibit the 3a channel by targeted drug repurposing. To that end, using three bacteria-based assays, we screened a library of 2839 approved-for-human-use drugs and identified the following potential channel-blockers: Capreomycin, Pentamidine, Spectinomycin, Kasugamycin, Plerixafor, Flumatinib, Litronesib, Darapladib, Floxuridine and Fludarabine. The stage is now set for examining the activity of these compounds in detailed electrophysiological studies and their impact on the whole virus with appropriate biosafety measures.

SARS-CoV-2 E protein is a potential ion channel that can be inhibited by Gliclazide and Memantine.

COVID-19 is one of the most impactful pandemics in recorded history. As such, the identification of inhibitory drugs against its etiological agent, SARS-CoV-2, is of utmost importance, and in particular, repurposing may provide the fastest route to curb the disease. As the first step in this route, we sought to identify an attractive and viable target in the virus for pharmaceutical inhibition. Using three bacteria-based assays that were tested on known viroporins, we demonstrate that one of its essential components, the E protein, is a potential ion channel and, therefore, is an excellent drug target. Channel activity was demonstrated for E proteins in other coronaviruses, providing further emphasis on the importance of this functionally to the virus' pathogenicity. The results of a screening effort involving a repurposing drug library of ion channel blockers yielded two compounds that inhibit the E protein: Gliclazide and Memantine. In conclusion, as a route to curb viral virulence and abate COVID-19, we point to the E protein of SARS-CoV-2 as an attractive drug target and identify off-label compounds that inhibit it.

The balance between side-chain and backbone-driven association in folding of the α-helical influenza A transmembrane peptide.

The correct balance between attractive, repulsive and peptide hydrogen bonding interactions must be attained for proteins to fold correctly. To investigate these important contributors, we sought a comparison of the folding between two 25-residues peptides, the influenza A M2 protein transmembrane domain (M2TM) and the 25-Ala (Ala25 ). M2TM forms a stable α-helix as is shown by circular dichroism (CD) experiments. Molecular dynamics (MD) simulations with adaptive tempering show that M2TM monomer is more dynamic in nature and quickly interconverts between an ensemble of various α-helical structures, and less frequently turns and coils, compared to one α-helix for Ala25 . DFT calculations suggest that folding from the extended structure to the α-helical structure is favored for M2TM compared with Ala25 . This is due to CH⋯O attractive interactions which favor folding to the M2TM α-helix, and cannot be described accurately with a force field. Using natural bond orbital (NBO) analysis and quantum theory atoms in molecules (QTAIM) calculations, 26 CH⋯O interactions and 22 NH⋯O hydrogen bonds are calculated for M2TM. The calculations show that CH⋯O hydrogen bonds, although individually weaker, have a cumulative effect that cannot be ignored and may contribute as much as half of the total hydrogen bonding energy, when compared to NH⋯O, to the stabilization of the α-helix in M2TM. Further, a strengthening of NH⋯O hydrogen bonding interactions is calculated for M2TM compared to Ala25 . Additionally, these weak CH⋯O interactions can dissociate and associate easily leading to the ensemble of folded structures for M2TM observed in folding MD simulations.

A model for the interaction between NF-kappa-B and ASPP2 suggests an I-kappa-B-like binding mechanism.

We used computational methods to study the interaction between two key proteins in apoptosis regulation: the transcription factor NF-kappa-B (NFkappaB) and the proapoptotic protein ASPP2. The C-terminus of ASPP2 contains ankyrin repeats and SH3 domains (ASPP2(ANK-SH3)) that mediate interactions with numerous apoptosis-related proteins, including the p65 subunit of NFkappaB (NFkappaB(p65)). Using peptide-based methods, we have recently identified the interaction sites between NFkappaB(p65) and ASPP2(ANK-SH3) (Rotem et al., J Biol Chem 283, 18990-18999). Here we conducted a computational study of protein docking and molecular dynamics to obtain a structural model of the complex between the full length proteins and propose a mechanism for the interaction. We found that ASPP2(ANK-SH3) binds two sites in NFkappaB(p65), at residues 236-253 and 293-313 that contain the nuclear localization signal (NLS). These sites also mediate the binding of NFkappaB to its natural inhibitor IkappaB, which also contains ankyrin repeats. Alignment of the ankyrin repeats of ASPP2(ANK-SH3) and IkappaB revealed that both proteins share highly similar interfaces at their binding sites to NFkappaB. Protein docking of ASPP2(ANK-SH3) and NFkappaB(p65), as well as molecular dynamics simulations of the proteins, provided structural models of the complex that are energetically similar to the NFkappaB-IkappaB determined structure. Our results show that ASPP2(ANK-SH3) binds NFkappaB(p65) in a similar manner to its natural inhibitor IkappaB, suggesting a possible novel role for ASPP2 as an NFkappaB inhibitor.

A trimerizing GxxxG motif is uniquely inserted in the severe acute respiratory syndrome (SARS) coronavirus spike protein transmembrane domain.

In an attempt to understand what distinguishes severe acute respiratory syndrome (SARS) coronavirus (SCoV) from other members of the coronaviridae, we searched for elements that are unique to its proteins and not present in any other family member. We identified an insertion of two glycine residues, forming the GxxxG motif, in the SCoV spike protein transmembrane domain (TMD), which is not found in any other coronavirus. This surprising finding raises an "oligomerization riddle": the GxxxG motif is a known dimerization signal, while the SCoV spike protein is known to be trimeric. Using an in vivo assay, we found that the SCoV spike protein TMD is oligomeric and that this oligomerization is driven by the GxxxG motif. We also found that the GxxxG motif contributes toward the trimerization of the entire spike protein; in that, mutations in the GxxxG motif decrease trimerization of the full-length protein expressed in mammalian cells. Using molecular modeling, we show that the SCoV spike protein TMD adopts a distinct and unique structure as opposed to all other coronaviruses. In this unique structure, the glycine residues of the GxxxG motif are facing each other, enhancing helix-helix interactions by allowing for the close positioning of the helices. This unique orientation of the glycine residues also stabilizes the trimeric bundle during multi-nanosecond molecular dynamics simulation in a hydrated lipid bilayer. To the best of our knowledge, this is the first demonstration that the GxxxG motif can potentiate other oligomeric forms beside a dimer. Finally, according to recent studies, the stabilization of the trimeric bundle is linked to a higher fusion activity of the spike protein, and the possible influence of the GxxxG motif on this feature is discussed.

Disorder influence on linear dichroism analyses of smectic phases.

Linear dichroism, the unequal absorption of parallel and perpendicular linear polarized light, is often used to determine the anisotropic ordering of rodlike polymers in a smectic phase, such as helices in a lipid bilayer. It is a measure of two properties of the sample: 1), orientation of the chromophore transition dipole moment (TDM) and 2), disorder. Since it is the orientation of the chromophore TDM that is needed for high resolution structural studies, it is imperative to either deconvolve sample disorder, or at a minimum, estimate its effect upon the calculated TDM orientation. Herein, a rigorous analysis of the effects of disorder is undertaken based on the recently developed Gaussian disorder model implemented in linear dichroism data. The calculation of both the rod tilt and rotational pitch angles as a function of the disorder and dichroism, yield the following conclusions: Disorders smaller than 5 degrees have a vanishingly small effect on the calculated polymer orientation, whereas values smaller than 10 degrees have a negligible effect on the calculated parameters. Disorders larger than 10 degrees have an appreciable effect on the calculated orientational parameters and as such must be estimated before any structural characterization. Finally the theory is tested on the HIV vpu transmembrane domain, employing experimental mosaicity measurements from x-ray reflectivity rocking scans and linear dichroism.

Distinct protein interfaces in transmembrane domains suggest an in vivo folding model.

Given the known high-resolution structures of alpha-helical transmembrane domains, we show that there are statistically distinct classes of transmembrane interfaces which relate to the folding and oligomerization of transmembrane domains. Distinct types of interfaces have been categorized and refer to those between: the same polypeptide chain, different polypeptide chains, helices that are sequential neighbors, and those that are nonsequential. These different interfaces may reflect different phases in the mechanism of transmembrane domain folding and are consistent with the current experimental evidence pertaining to the folding and oligomerization of transmembrane domains. The classes of helix-helix interfaces have been identified in terms of the numbers and different types of pairwise amino acid interactions. The specific measures used are interaction entropy, the information content of interacting partners compared to a random set of contacts, the amino acid composition of the classes and the abundances of specific amino acid pairs in close contact. Knowledge of the clear differences in the types of helix-helix contacts helps with the derivation of knowledge-based constraints which until now have focused on only the interiors of transmembrane domains as compared to the exterior. Taken together, an in vivo model for membrane protein folding is presented, which is distinct from the familiar two-stage model. The model takes into account the different interfaces of membrane helices defined herein, and the available data regarding folding in the translocation channel.

A highly unusual palindromic transmembrane helical hairpin formed by SARS coronavirus E protein.

The agent responsible for the recent severe acute respiratory syndrome (SARS) outbreak is a previously unidentified coronavirus. While there is a wealth of epidemiological studies, little if any molecular characterization of SARS coronavirus (SCoV) proteins has been carried out. Here we describe the molecular characterization of SCoV E protein, a critical component of the virus responsible for virion envelope morphogenesis. We conclusively show that SCoV E protein contains an unusually short, palindromic transmembrane helical hairpin around a previously unidentified pseudo-center of symmetry, a structural feature which seems to be unique to SCoV. The hairpin deforms lipid bilayers by way of increasing their curvature, providing for the first time a molecular explanation of E protein's pivotal role in viral budding. The molecular understanding of this critical component of SCoV may represent the beginning of a concerted effort aimed at inhibiting its function, and consequently, viral infectivity.

Site-specific vibrational dynamics of the CD3zeta membrane peptide using heterodyned two-dimensional infrared photon echo spectroscopy.

Heterodyned two-dimensional infrared (2D IR) spectroscopy has been used to study the amide I vibrational dynamics of a 27-residue peptide in lipid vesicles that encompasses the transmembrane domain of the T-cell receptor CD3zeta. Using 1-(13)C[Double Bond](18)O isotope labeling, the amide I mode of the 49-Leucine residue was spectroscopically isolated and the homogeneous and inhomogeneous linewidths of this mode were measured by fitting the 2D IR spectrum collected with a photon echo pulse sequence. The pure dephasing and inhomogeneous linewidths are 2 and 32 cm(-1), respectively. The population relaxation time of the amide I band was measured with a transient grating, and it contributes 9 cm(-1) to the linewidth. Comparison of the 49-Leucine amide I mode and the amide I band of the entire CD3zeta peptide reveals that the vibrational dynamics are not uniform along the length of the peptide. Possible origins for the large amount of inhomogeneity present at the 49-Leucine site are discussed.

Modeling the structure of the respiratory syncytial virus small hydrophobic protein by silent-mutation analysis of global searching molecular dynamics.

Human respiratory syncytial virus (RSV) encodes a small hydrophobic (SH) protein, whose function in the life cycle of the virus is unknown. Recent channel activity measurements of the protein suggest that like other viroporins, SH may assemble into a homo-oligomeric ion channel. To further our understanding of this potentially important protein, a new strategy was implemented in order to model the transmembrane oligomeric bundle of the protein. Global searching molecular dynamic simulations of SH proteins from eight different viral strains, each at different oligomeric states, as well as different lengths of the putative transmembrane domain, were undertaken. Taken together, a total of 45 different global molecular dynamic simulations pointed to a single pentameric structure for the protein that was found in all of the different variants. The model of the structure obtained is a channel-like homopentamer whose minimal transmembrane pore diameter is 1.46 A.

Syntaxin 1A modulates the voltage-gated L-type calcium channel (Ca(v)1.2) in a cooperative manner.

Syntaxin 1A (Sx1A) modifies the activity of voltage-gated Ca2+ channels acting via the cytosolic and the two vicinal cysteines (271 and 272) at the transmembrane domain. Here we show that Sx1A modulates the Lc-type Ca2+ channel, Cav1.2, in a cooperative manner, and we explore whether channel clustering or the Sx1A homodimer is responsible for this activity. Sx1A formed homodimers but, when mutated at the two vicinal transmembrane domain cysteines, was unable to either dimerize or modify the channel activity suggesting disulfide bond formation. Moreover, applying global molecular dynamic search established a theoretical prospect of generating a disulfide bond between two Sx1A transmembrane helices. Nevertheless, Sx1A activity was not correlated with Sx1A homodimer. Application of a vicinal thiol reagent, phenylarsine oxide, abolished Sx1A action indicating the accessibility of Cys-271,272 thiols. Sx1A inhibition of channel activity was restored by phenylarsine oxide antidote, 2,3-dimercaptopropanol, consistent with thiol interaction of Sx1A. In addition, the supralinear mode of channel inhibition was correlated to the monomeric form of Sx1A and was apparent only when the three channel subunits alpha11.2/alpha2delta1/beta2a were present. This functional demonstration of cooperativity suggests that the three-subunit channel responds as a cluster, and Sx1A monomers associate with a dimer (or more) of a three-subunit Ca2+ channel. Consistent with channel cluster linked to Sx1A, a conformational change driven by membrane depolarization and Ca2+ entry would rapidly be transduced to the exocytotic machinery. As shown herein, the supralinear relationship between Sx1A and the voltage-gated Ca2+ channel within the cluster could convey the cooperativity that distinguishes the process of neurotransmitter release.

Hydrogen/deuterium exchange of hydrophobic peptides in model membranes by electrospray ionization mass spectrometry.

We demonstrate here that the hydrogen/deuterium solvent exchange (HDX) properties of the transmembrane fragment of the M2 protein of Influenza A (M2-TM) incorporated into lipid vesicles or detergent micelles can be studied with straightforward electrospray (ESI) and nanospray mass spectrometry (MS) configurations provided that key factors, including sample preparation techniques, are optimized. Small unilamellar vesicle preparations were obtained by solubilizing dimyristoyl phosphatidylcholine (DMPC) and the M2-TM peptide in aqueous solution with n-octyl-beta-D-glycopyranoside, followed by dialysis to remove the detergent. Electron microscopy experiments revealed that subsequent concentration by centrifugation introduced large multilamellar aggregates that were not compatible with ESI-MS. By contrast, a lyophilization-based concentration procedure, followed by thawing above the liquid crystal transition temperature of the lipid component, maintained the liposome size profile and yielded excellent ion fluxes in both ESI-MS and nano-ESI-MS. Using these methods the global HDX profile of M2-TM in aqueous DMPC vesicles was compared with that in methanol, demonstrating that several amide sites were protected from exchange by the lipid membrane. We also show that hydrophobic peptides can be detected by ESI-MS in the presence of a large molar excess of the detergent Triton X-100. The rate of HDX of M2-TM in Triton X-100 micelles was faster than that in DMPC vesicles but slower than when the peptide had been denatured in methanol. These results indicate that the accessibility of backbone amide sites to the solvent can be profoundly affected by membrane protein structure and dynamics, as well as the properties of model bilayer systems.