Viroporins of Mpox Virus.

Mpox or monkeypox virus (MPXV) belongs to the subclass of Poxviridae and has emerged recently as a global threat. With a limited number of anti-viral drugs available for this new virus species, it is challenging to thwart the illness it begets. Therefore, characterizing new drug targets in the virus may prove advantageous to curbing the disease. Since channels as a family are excellent drug targets, we have sought to identify viral ion channels for this virus, which are instrumental in formulating channel-blocking anti-viral drugs. Bioinformatics analyses yielded eight transmembranous proteins smaller or equal to 100 amino acids in length. Subsequently, three independent bacteria-based assays have pointed to five of the eight proteins that exhibit ion channel activity. Finally, we propose a tentative structure of four ion channels from their primary amino acid sequences, employing AlphaFold2 and molecular dynamic simulation methods. These results may represent the first steps in characterizing MPXV viroporins en route to developing blockers that inhibit their function.

Quantitative Analysis of Multiplex H-Bonds.

H-bonding is the predominant geometrical determinant of biomolecular structure and interactions. As such, considerable analyses have been undertaken to study its detailed energetics. The focus, however, has been mostly reserved for H-bonds comprising a single donor and a single acceptor. Herein, we measure the prevalence and energetics of multiplex H-bonds that are formed between three or more groups. We show that 92% of all transmembrane helices have at least one non-canonical H-bond formed by a serine or threonine residue whose hydroxyl side chain H-bonds to an over-coordinated carbonyl oxygen at position i-4, i-3, or i in the sequence. Isotope-edited FTIR spectroscopy, coupled with DFT calculations, enables us to determine the bond enthalpies, pointing to values that are up to 127% higher than that of a single canonical H-bond. We propose that these strong H-bonds serve to stabilize serine and threonine residues in hydrophobic environments while concomitantly providing them flexibility between different configurations, which may be necessary for function.

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 Robust Proton Flux (pHlux) Assay for Studying the Function and Inhibition of the Influenza A M2 Proton Channel.

The M2 protein is an important target for drugs in the fight against the influenza virus. Because of the emergence of resistance against antivirals directed toward the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays for testing potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In this work, we describe a fluorescence sensor-based assay, which we termed "pHlux", that measures proton conduction through M2 when synthesized from an expression vector in Escherichia coli. The assay was compared to a previously established bacterial potassium ion transport complementation assay, and the results were compared to simulations obtained from analysis of a computational model of M2 and its interaction with inhibitor molecules. The inhibition of M2 was measured for five different inhibitors, including Rimantadine, Amantadine, and spiro type compounds, and the drug resistance of the M2 mutant variants (swine flu, V27A, and S31N) was confirmed. We demonstrate that the pHlux assay is robust and highly sensitive and shows potential for high-throughput screening.

Random Mutagenesis Analysis of the Influenza A M2 Proton Channel Reveals Novel Resistance Mutants.

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M_089 (N13I, I35L, and F47L) and M_272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

Strength of a bifurcated H bond.

Macromolecules are characterized by their particular arrangement of H bonds. Many of these interactions involve a single donor and acceptor pair, such as the regular H-bonding pattern between carbonyl oxygens and amide H(+)s four residues apart in α-helices. The H-bonding potential of some acceptors, however, leads to the phenomenon of overcoordination between two donors and one acceptor. Herein, using isotope-edited Fourier transform infrared measurements and density functional theory (DFT) calculations, we measured the strength of such bifurcated H bonds in a transmembrane α-helix. Frequency shifts of the (13)C=(18)O amide I mode were used as a reporter of the strength of the bifurcated H bond from a thiol and hydroxyl H(+) at residue i + 4. DFT calculations yielded very similar frequency shifts and an energy of -2.6 and -3.4 kcal/mol for the thiol and hydroxyl bifurcated H bonds, respectively. The strength of the intrahelical bifurcated H bond is consistent with its prevalence in hydrophobic environments and is shown to significantly impact side-chain rotamer distribution.

Computational and experimental analysis of drug binding to the Influenza M2 channel.

The Influenza Matrix 2 (M2) protein is the target of Amantadine and Rimantadine which block its H(+) channel activity. However, the potential of these aminoadamantyls to serve as anti-flu agents is marred by the rapid resistance that the virus develops against them. Herein, using a cell based assay that we developed, we identify two new aminoadamantyl derivatives that show increased activity against otherwise resistant M2 variants. In order to understand the distinguishing binding patterns of the different blockers, we computed the potential of mean force of the drug binding process. The results reveal that the new derivatives are less mobile and bind to a larger pocket in the channel. Finally, such analyses may prove useful in designing new, more effective M2 blockers as a means of curbing influenza. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.

Mechanistic studies of the apical sodium-dependent bile acid transporter.

In mammals, the apical sodium-dependent bile acid transporter (ASBT) is responsible for the reuptake of bile acid from the intestine, thus recycling bile acid that is secreted from the gallbladder, for the purpose of digestion. As bile acid is synthesized from cholesterol, ASBT inhibition could have important implications in regulation of cholesterol levels in the blood. We report on a simulation study of the recently resolved structures of the inward-facing ASBT from Neisseria meningitidis and from Yersinia frederiksenii, as well as of an ASBT variant from Yersinia frederiksenii suggested to be in the outward-facing conformation. Classical and steered atomistic simulations and comprehensive potential of mean force analyses of ASBT, both in the absence and presence of ions and substrate, allow us to characterize and gain structural insights into the Na(+) binding sites and propose a mechanistic model for the transport cycle. In particular, we investigate structural features of the ion translocation pathway, and suggest a third putative Na(+) binding site. Our study sheds light on the structure-function relationship of bacterial ASBT and may promote a deeper understanding of transport mechanism altogether.

Gaining insight into membrane protein structure using isotope-edited FTIR.

FTIR spectroscopy has long been used as a tool used to gain average structural information on proteins. With the advent of stable isotope editing, FTIR can be used to derive accurate information on isolated amino acids. In particular, in an anisotropic sample such as membrane layers, it is possible to measure the orientation of the peptidic carbonyl groups. Herein, we review the theory that enables one to obtain accurate restraints from FTIR spectroscopy, alongside considerations for sample suitability and general applicability. We also propose approaches that may be used to generate structural models of simple membrane proteins based on FTIR orientational restraints. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

Self-interaction of transmembrane helices representing pre-clusters from the human single-span membrane proteins.

MOTIVATION: Most integral membrane proteins form dimeric or oligomeric complexes. Oligomerization is frequently supported by the non-covalent interaction of transmembrane helices. It is currently not clear how many high-affinity transmembrane domains (TMD) exist in a proteome and how specific their interactions are with respect to preferred contacting faces and their underlying residue motifs. RESULTS: We first identify a threshold of 55% sequence similarity, which demarcates the border between meaningful alignments of TMDs and chance alignments. Clustering the human single-span membrane proteome using this threshold groups ~40% of the TMDs. The homotypic interaction of the TMDs representing the 33 largest clusters was systematically investigated under standardized conditions. The results reveal a broad distribution of relative affinities. High relative affinity frequently coincides with (i) the existence of a preferred helix-helix interface and (ii) sequence specificity as indicated by reduced affinity after mutating conserved residues. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Lysine tRNA fragments and miR-194-5p co-regulate hepatic steatosis via ß-Klotho and perilipin 2.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) involves hepatic accumulation of intracellular lipid droplets via incompletely understood processes. Here, we report distinct and cooperative NAFLD roles of LysTTT-5'tRF transfer RNA fragments and microRNA miR-194-5p. METHODS: Combined use of diet induced obese mice with human-derived oleic acid-exposed Hep G2 cells revealed new NAFLD roles of LysTTT-5'tRF and miR-194-5p. RESULTS: Unlike lean animals, dietary-induced NAFLD mice showed concurrent hepatic decrease of both LysTTT-5'tRF and miR-194-5p levels, which were restored following miR-132 antisense oligonucleotide treatment which suppresses hepatic steatosis. Moreover, exposing human-derived Hep G2 cells to oleic acid for 7 days co-suppressed miR-194-5p and LysTTT-5'tRF levels while increasing lipid accumulation. Inversely, transfecting fattened cells with a synthetic LysTTT-5'tRF mimic elevated mRNA levels of the metabolic regulator ß-Klotho while decreasing triglyceride amounts by 30% within 24 h. In contradistinction, antisense suppression of miR-194-5p induced accumulation of its novel target, the NAFLD-implicated lipid droplet-coating PLIN2 protein. Further, two out of 15 steatosis-alleviating screened drug-repurposing compounds, Danazol and Latanoprost, elevated miR-194-5p or LysTTT-5'tRF levels. CONCLUSION: Our findings highlight the different yet complementary roles of miR-194-5p and LysTTT-5'tRF and offer new insights into the complex roles of small non-coding RNAs and the multiple pathways involved in NAFLD pathogenesis.

Exhaustive mutational analysis of severe acute respiratory syndrome coronavirus 2 ORF3a: An essential component in the pathogen's infectivity cycle.

Detailed knowledge of a protein's key residues may assist in understanding its function and designing inhibitors against it. Consequently, such knowledge of one of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)'s proteins is advantageous since the virus is the etiological agent behind one of the biggest health crises of recent times. To that end, we constructed an exhaustive library of bacteria differing from each other by the mutated version of the virus's ORF3a viroporin they harbor. Since the protein is harmful to bacterial growth due to its channel activity, genetic selection followed by deep sequencing could readily identify mutations that abolish the protein's function. Our results have yielded numerous mutations dispersed throughout the sequence that counteract ORF3a's ability to slow bacterial growth. Comparing these data with the conservation pattern of ORF3a within the coronavirinae provided interesting insights: Deleterious mutations obtained in our study corresponded to conserved residues in the protein. However, despite the comprehensive nature of our mutagenesis coverage (108 average mutations per site), we could not reveal all of the protein's conserved residues. Therefore, it is tempting to speculate that our study unearthed positions in the protein pertinent to channel activity, while other conserved residues may correspond to different functionalities of ORF3a. In conclusion, our study provides important information on a key component of SARS-CoV-2 and establishes a procedure to analyze other viroporins comprehensively.

Resistance characteristics of influenza to amino-adamantyls.

The recent outbreaks of avian flu in Southeast Asia and swine flu in Mexico City painfully exemplify the ability of the influenza virus to rapidly mutate and develop resistance to modern medicines. This review seeks to detail the molecular mechanism by which the influenza virus has obtained resistance to amino-adamantyls, one of only two classes of drugs that combat the flu. Amino-adamantyls target the viral M2 H(+) channel and have become largely ineffective due to mutations in the transmembrane domain of the protein. Herein we describe these resistance rendering mutations and the compounded effects they have upon the protein's function and resulting virus viability.

Quantitative analysis of influenza M2 channel blockers.

The influenza M2 H(+) channel enables the concomitant acidification of the viral lumen upon endosomic internalization. This process is critical to the viral infectivity cycle, demonstrated by the fact that M2 is one of only two targets for anti-flu agents. However, aminoadamantyls that block the M2 channel are of limited therapeutic use due to the emergence of resistance mutations in the protein. Herein, using an assay that involves expression of the protein in Escherichia coli with resultant growth retardation, we present quantitative measurements of channel blocker interactions. Comparison of detailed K(s) measurements of different drugs for several influenza channels, shows that the swine flu M2 exhibits the highest resistance to aminoadamantyls of any channel known to date. From the perspective of the blocker, we show that rimantadine is consistently a better blocker of M2 than amantadine. Taken together, such detailed and quantitative analyses provide insight into the mechanism of this important and pharmaceutically relevant channel blocker system.

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.

Targeting Viral Ion Channels: A Promising Strategy to Curb SARS-CoV-2.

SARS-CoV-2 is the etiological agent COVID-19, one of the most impactful health crises afflicting humanity in recent decades. While research advances have yielded several treatment and prevention options, the pandemic is slow to abate, necessitating an expansion of our treatment arsenal. As a member of the coronaviridae, SARS-CoV-2 contains several ion channels, of which E and 3a are the best characterized. Since ion channels as a family are excellent drug targets, we sought to inhibit both viroporins as a means to curb infectivity. In a previous targeted study, we identified several blockers to each channel from an extensive drug repurposing library. Herein, we examined the ability of said compounds on the whole virus in cellulo. Gratifyingly, many of the blockers exhibited antiviral activity in a stringent assay examining protection from viral-driven death. In particular, darapladib and flumatinib, both 3a blockers, displayed potent antiviral activity. Furthermore, appreciable synergism between flumatinib and several E blockers was identified in a concentration regime in which the compounds are present in human plasma following oral administration. Taken together, targeting ion channels represents a promising approach to both augment and complement our antiviral arsenal against COVID-19.

Zika M-A Potential Viroporin: Mutational Study and Drug Repurposing.

Genus Flavivirus contains several important human pathogens. Among these, the Zika virus is an emerging etiological agent that merits concern. One of its structural proteins, prM, plays an essential role in viral maturation and assembly, making it an attractive drug and vaccine development target. Herein, we have characterized ZikV-M as a potential viroporin candidate using three different bacteria-based assays. These assays were subsequently employed to screen a library of repurposed drugs from which ten compounds were identified as ZikV-M blockers. Mutational analyses of conserved amino acids in the transmembrane domain of other flaviviruses, including West Nile and Dengue virus, were performed to study their role in ion channel activity. In conclusion, our data show that ZikV-M is a potential ion channel that can be used as a drug target for high throughput screening and drug repurposing.

How do aminoadamantanes block the influenza M2 channel, and how does resistance develop?

The interactions between channels and their cognate blockers are at the heart of numerous biomedical phenomena. Herein, we unravel one particularly important example bearing direct pharmaceutical relevance: the blockage mechanism of the influenza M2 channel by the anti-flu amino-adamantyls (amantadine and rimantadine) and how the channel and, consequently, the virus develop resistance against them. Using both computational analyses and experimental verification, we find that amino-adamantyls inhibit M2's H(+) channel activity by electrostatic hindrance due to their positively charged amino group. In contrast, the hydrophobic adamantyl moiety on its own does not impact conductivity. Additionally, we were able to uncover how mutations in M2 are capable of retaining drug binding on the one hand yet rendering the protein and the mutated virus resistant to amino-adamantyls on the other hand. We show that the mutated, drug-resistant protein has a larger binding pocket for the drug. Hence, despite binding the channel, the drug remains sufficiently mobile so as not to exert a H(+)-blocking positive electrostatic hindrance. Such insight into the blocking mechanism of amino-adamantyls, and resistance thereof, may aid in the design of next-generation anti-flu agents.

Dynamic control of slow water transport by aquaporin 0: implications for hydration and junction stability in the eye lens.

Aquaporin 0 (AQP0), the most abundant membrane protein in mammalian lens fiber cells, not only serves as the primary water channel in this tissue but also appears to mediate the formation of thin junctions between fiber cells. AQP0 is remarkably less water permeable than other aquaporins, but the structural basis and biological significance of this low permeability remain uncertain, as does the permeability of the protein in a reported junctional form. To address these issues, we performed molecular dynamics (MD) simulations of water transport through membrane-embedded AQP0 in both its (octameric) junctional and (tetrameric) nonjunctional forms. From our simulations, we measured an osmotic permeability for the nonjunctional form that agrees with experiment and found that the distinct dynamics of the conserved, lumen-protruding side chains of Tyr-23 and Tyr-149 modulate water passage, accounting for the slow permeation. The junctional and nonjunctional forms conducted water equivalently, in contrast to a previous suggestion based on static crystal structures that water conduction is lost on junction formation. Our analysis suggests that the low water permeability of AQP0 may help maintain the mechanical stability of the junction. We hypothesize that the structural features leading to low permeability may have evolved in part to allow AQP0 to form junctions that both conduct water and contribute to the organizational structure of the fiber cell tissue and microcirculation within it, as required to maintain transparency of the lens.