14-3-3 interaction with phosphodiesterase 8A sustains PKA signaling and downregulates the MAPK pathway.

The cAMP/PKA and mitogen-activated protein kinase (MAPK) signaling cascade control many cellular processes and are highly regulated for optimal cellular responses upon external stimuli. Phosphodiesterase 8A (PDE8A) is an important regulator that inhibits signaling via cAMP-dependent PKA by hydrolyzing intracellular cAMP pool. Conversely, PDE8A activates the MAPK pathway by protecting CRAF/Raf1 kinase from PKA-mediated inhibitory phosphorylation at Ser259 residue, a binding site of scaffold protein 14-3-3. It still remains enigmatic as to how the cross-talk involving PDE8A regulation influences cAMP/PKA and MAPK signaling pathways. Here, we report that PDE8A interacts with 14-3-3ζ in both yeast and mammalian system, and this interaction is enhanced upon the activation of PKA, which phosphorylates PDE8A's Ser359 residue. Biophysical characterization of phospho-Ser359 peptide with 14-3-3ζ protein further supports their interaction. Strikingly, 14-3-3ζ reduces the catalytic activity of PDE8A, which upregulates the cAMP/PKA pathway while the MAPK pathway is downregulated. Moreover, 14-3-3ζ in complex with PDE8A and cAMP-bound regulatory subunit of PKA, RIα, delays the deactivation of PKA signaling. Our results define 14-3-3ζ as a molecular switch that operates signaling between cAMP/PKA and MAPK by associating with PDE8A.

Cryo-Electron Microscopy Structure of the Mycobacterium tuberculosis Cytochrome bcc:aa3 Supercomplex and a Novel Inhibitor Targeting Subunit Cytochrome cI.

The mycobacterial cytochrome bcc:aa3 complex deserves the name "supercomplex" since it combines three cytochrome oxidases-cytochrome bc, cytochrome c, and cytochrome aa3-into one supramolecular machine and performs electron transfer for the reduction of oxygen to water and proton transport to generate the proton motive force for ATP synthesis. Thus, the bcc:aa3 complex represents a valid drug target for Mycobacterium tuberculosis infections. The production and purification of an entire M. tuberculosis cytochrome bcc:aa3 are fundamental for biochemical and structural characterization of this supercomplex, paving the way for new inhibitor targets and molecules. Here, we produced and purified the entire and active M. tuberculosis cyt-bcc:aa3 oxidase, as demonstrated by the different heme spectra and an oxygen consumption assay. The resolved M. tuberculosis cyt-bcc:aa3 cryo-electron microscopy structure reveals a dimer with its functional domains involved in electron, proton, oxygen transfer, and oxygen reduction. The structure shows the two cytochrome cIcII head domains of the dimer, the counterpart of the soluble mitochondrial cytochrome c, in a so-called "closed state," in which electrons are translocated from the bcc to the aa3 domain. The structural and mechanistic insights provided the basis for a virtual screening campaign that identified a potent M. tuberculosis cyt-bcc:aa3 inhibitor, cytMycc1. cytMycc1 targets the mycobacterium-specific α3-helix of cytochrome cI and interferes with oxygen consumption by interrupting electron translocation via the cIcII head. The successful identification of a new cyt-bcc:aa3 inhibitor demonstrates the potential of a structure-mechanism-based approach for novel compound development.

Structure activity relationship of pyrazinoic acid analogs as potential antimycobacterial agents.

Tuberculosis (TB) remains a leading cause of infectious disease-related mortality and morbidity. Pyrazinamide (PZA) is a critical component of the first-line TB treatment regimen because of its sterilizing activity against non-replicating Mycobacterium tuberculosis (Mtb), but its mechanism of action has remained enigmatic. PZA is a prodrug converted by pyrazinamidase encoded by pncA within Mtb to the active moiety, pyrazinoic acid (POA) and PZA resistance is caused by loss-of-function mutations to pyrazinamidase. We have recently shown that POA induces targeted protein degradation of the enzyme PanD, a crucial component of the coenzyme A biosynthetic pathway essential in Mtb. Based on the newly identified mechanism of action of POA, along with the crystal structure of PanD bound to POA, we designed several POA analogs using structure for interpretation to improve potency and overcome PZA resistance. We prepared and tested ring and carboxylic acid bioisosteres as well as 3, 5, 6 substitutions on the ring to study the structure activity relationships of the POA scaffold. All the analogs were evaluated for their whole cell antimycobacterial activity, and a few representative molecules were evaluated for their binding affinity, towards PanD, through isothermal titration calorimetry. We report that analogs with ring and carboxylic acid bioisosteres did not significantly enhance the antimicrobial activity, whereas the alkylamino-group substitutions at the 3 and 5 position of POA were found to be up to 5 to 10-fold more potent than POA. Further development and mechanistic analysis of these analogs may lead to a next generation POA analog for treating TB.

Targeting the menaquinol binding loop of mycobacterial cytochrome bd oxidase.

Mycobacteria have shown enormous resilience to survive and persist by remodeling and altering metabolic requirements. Under stringent conditions or exposure to drugs, mycobacteria have adapted to rescue themselves by shutting down their major metabolic activity and elevate certain survival factor levels and efflux pathways to survive and evade the effects of drug treatments. A fundamental feature in this adaptation is the ability of mycobacteria to vary the enzyme composition of the electron transport chain (ETC), which generates the proton motive force for the synthesis of adenosine triphosphate via oxidative phosphorylation. Mycobacteria harbor dehydrogenases to fuel the ETC, and two terminal respiratory oxidases, an aa3-type cytochrome c oxidase (cyt-bcc-aa3) and a bacterial specific cytochrome bd-type menaquinol oxidase (cyt-bd). In this study, we employed homology modeling and structure-based virtual screening studies to target mycobacteria-specific residues anchoring the b558 menaquinol binding region of Mycobacterium tuberculosis cyt-bd oxidase to obtain a focused library. Furthermore, ATP synthesis inhibition assays were carried out. One of the ligands MQL-H2 inhibited both NADH2- and succinate-driven ATP synthesis inhibition of Mycobacterium smegmatis inside-out vesicles in micromolar potency. Similarly, MQL-H2 also inhibited NADH2-driven ATP synthesis in inside-out vesicles of the cytochrome-bcc oxidase deficient M. smegmatis strain. Since neither varying the electron donor substrates nor deletion of the cyt-bcc oxidase, a major source of protons, hindered the inhibitory effects of the MQL-H2, reflecting that MQL-H2 targets the terminal oxidase cytochrome bd oxidase, which was consistent with molecular docking studies. Characterization of novel cytochrome bd oxidase Menaquinol binding domain inhibitor (MQL-H2) using virtual screening and ATP synthesis inhibition assays.

Discovery of a Novel Mycobacterial F-ATP Synthase Inhibitor and its Potency in Combination with Diarylquinolines.

The F1 FO -ATP synthase is required for growth and viability of Mycobacterium tuberculosis and is a validated clinical target. A mycobacterium-specific loop of the enzyme's rotary γ subunit plays a role in the coupling of ATP synthesis within the enzyme complex. We report the discovery of a novel antimycobacterial, termed GaMF1, that targets this γ subunit loop. Biochemical and NMR studies show that GaMF1 inhibits ATP synthase activity by binding to the loop. GaMF1 is bactericidal and is active against multidrug- as well as bedaquiline-resistant strains. Chemistry efforts on the scaffold revealed a dynamic structure activity relationship and delivered analogues with nanomolar potencies. Combining GaMF1 with bedaquiline or novel diarylquinoline analogues showed potentiation without inducing genotoxicity or phenotypic changes in a human embryonic stem cell reporter assay. These results suggest that GaMF1 presents an attractive lead for the discovery of a novel class of anti-tuberculosis F-ATP synthase inhibitors.

Combination of pharmacophore hypothesis and molecular docking to identify novel inhibitors of HCV NS5B polymerase.

Hepatitis C virus (HCV) infection or HCV-related liver diseases are now shown to cause more than 350,000 deaths every year. Adaptability of HCV genome to vary its composition and the existence of multiple strains makes it more difficult to combat the emergence of drug-resistant HCV infections. Among the HCV polyprotein which has both the structural and non-structural regions, the non-structural protein NS5B RNA-dependent RNA polymerase (RdRP) mainly mediates the catalytic role of RNA replication in conjunction with its viral protein machinery as well as host chaperone proteins. Lack of such RNA-dependent RNA polymerase enzyme in host had made it an attractive and hotly pursued target for drug discovery efforts. Recent drug discovery efforts targeting HCV RdRP have seen success with FDA approval for sofosbuvir as a direct-acting antiviral against HCV infection. However, variations in drug-binding sites induce drug resistance, and therefore targeting allosteric sites could delay the emergence of drug resistance. In this study, we focussed on allosteric thumb site II of the non-structural protein NS5B RNA-dependent RNA polymerase and developed a five-feature pharmacophore hypothesis/model which estimated the experimental activity with a strong correlation of 0.971 & 0.944 for training and test sets, respectively. Further, the Güner-Henry score of 0.6 suggests that the model was able to discern the active and inactive compounds and enrich the true positives during a database search. In this study, database search and molecular docking results supported by experimental HCV viral replication inhibition assays suggested ligands with best fitness to the pharmacophore model dock to the key residues involved in thumbs site II, which inhibited the HCV 1b viral replication in sub-micro-molar range.

NMR solution structure of C2 domain of MFG-E8 and insights into its molecular recognition with phosphatidylserine.

MFG-E8 (also known as lactadherin), which is a secreted glycoprotein from a variety of cell types, possesses two EGF domains and tandem C domains with sequence homology to that of blood coagulation proteins factor V and factor VIII. MFG-E8 binds to phosphatidylserine (PS) in membranes with high affinity. We have recently shown that the C2 domain of MFG-E8 bears more specificity toward PS when compared with phosphatidylcholine (PC), another phospholipid thought to be involved in the immune function of phagocytes. In our current study, we have determined the solution structure of the C2 domain by nuclear magnetic resonance (NMR) spectroscopy, and characterized the molecular basis of binding between the C2 domain and PS by (31)P-NMR spectroscopy. Furthermore, we also verified that that positively charged and aromatic residues clustered in loops 1-3 of the C2 domain play key roles in recognizing PS in apoptotic cells.

Crystal structure of Plasmodium vivax FK506-binding protein 25 reveals conformational changes responsible for its noncanonical activity.

The malarial parasites currently remain one of the most dreadful parasites, which show increasing trend of drug resistance to the currently available antimalarial drugs. Thus, the need to identify and characterize new protein targets in these parasites can aid to design novel therapeutic strategies to combat malaria. Recently, the conserved FK506-binding protein family members with molecular weight of 35 kDa from Plasmodium falciparum and Plasmodium vivax (referred to as PfFKBP35 and PvFKBP35, respectively) were identified for drug targeting. Further data mining revealed a 25-kDa FKBP (FKBP25) family member present in the parasites. FKBP25 belongs to a unique class of FKBP, because it is a nuclear FKBP with multiple protein-binding partners. Apart from immune regulation, it is also known for its chaperoning role in various cellular processes such as transcription regulation and trafficking. Here, we present the biochemical characterization and 1.9-Å crystal structure of an N-terminal truncated FKBP25 from P. vivax (PvFKBP25(72-209)). The protein reveals the noncanonical nature with unique structural changes observed in the loops flanking the active site, concealing the binding pocket. Further, a potential calmodulin-binding domain, which is absent in human FKBP25, is observed in this protein. Although the functional implication of Plasmodium FKBP25 in malaria still remains elusive, we speculate that the notable conformational changes in its structure might serve as an overture in understanding its molecular mechanism.

The flavonoid myricetin reduces nocturnal melatonin levels in the blood through the inhibition of serotonin N-acetyltransferase.

Melatonin is secreted during the hours of darkness and is thought to influence the circadian and seasonal timing of a variety of physiological processes. AANAT, which is expressed in the pineal gland, retina, and various other tissues, catalyzes the conversion of serotonin to N-acetylserotonin and is the rate-limiting enzyme in the biosynthetic pathway of melatonin. The compounds that modulate the activity of AANAT can be used to treat patients with circadian rhythm disorders that are associated with specific circadian rhythm alterations, such as shift work disorder. In the present study, we screened modulators of AANAT activity from the water extracts of medicinal plants. Among the 267 tested medicinal plant extracts, Myricae Cortex (Myrica rubra), Perillae Herba (Perilla sikokiana), and Eriobotryae Folium (Eriobotrya japonica) showed potent inhibition of AANAT activity. Myricetin (5,7,3',4',5'-pentahydroxyflavonol), a main component of the Myricae Cortex, strongly inhibited the activity of AANAT and probably block the access to the substrate by docking to the catalytic residues that are important for AANAT activity. Myricetin significantly decreased the nocturnal serum melatonin levels in rats. In addition, the locomotor activity of rats treated with myricetin decreased during the nighttime and slightly increased throughout the day. These results suggest that myricetin could be used as a therapy to increase nighttime alertness by changing the circadian rhythm of serum melatonin and locomotor activity.

Novel targets and inhibitors of the Mycobacterium tuberculosis cytochrome bd oxidase to foster anti-tuberculosis drug discovery.

INTRODUCTION: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is the most devastating bacterial disease. Multidrug-resistant Mtb strains are spreading worldwide, underscoring the need for new anti-TB targets and inhibitors. The respiratory chain complexes, including the cytochrome bd oxidase (cyt-bd), have been identified as an attractive target for drug development. Recent novel structural and mechanistic insight as well as inhibitors of Mtb's cyt-bd brought this enzyme into the focus. AREAS COVERED: In this review, the authors describe conditions that stimulate the biogenesis of Mtb cyt-bd, its structural-, mechanistic-, and substrate-binding traits. They discuss the present Mtb cyt-bd inhibitors, novel targets within the enzyme and structure activity relationship features that are required for mycobacterial cyt-bd inhibition and augment their understanding on improving the potency of cyt-bd inhibitors. EXPERT OPINION: A deeper structure-mechanistic understanding of Mtb's cyt-bd is a prerequisite for in silico efforts to: (i) identify pathogen specific targets for the design of novel nontoxic hit molecules, forming the platform for the development of new leads, (ii) design mechanism of action studies, (iii) perform medicinal chemistry of existing inhibitors to improve their potency and pharmacokinetic/-dynamic properties. Phase studies with such optimized cyt-bd inhibitors in combination with anti-TB compounds targeting the oxidative phosphorylation pathway is recommended.

Coomassie brilliant blue G-250 acts as a potential chemical chaperone to stabilize therapeutic insulin.

Our studies show Coomassie Brilliant Blue G-250 as a promising chemical chaperone that stabilises the α-helical native human insulin conformers, disrupting their aggregation. Furthermore, it also increases the insulin secretion. This multipolar effect coupled with its non-toxic nature could be useful for developing highly bioactive, targeted and biostable therapeutic insulin.

GaMF1.39's antibiotic efficacy and its enhanced antitubercular activity in combination with clofazimine, Telacebec, ND-011992, or TBAJ-876.

IMPORTANCE: New drugs are needed to combat multidrug-resistant tuberculosis. The electron transport chain (ETC) maintains the electrochemical potential across the cytoplasmic membrane and allows the production of ATP, the energy currency of any living cell. The mycobacterial engine F-ATP synthase catalyzes the formation of ATP and has come into focus as an attractive and rich drug target. Recent deep insights into these mycobacterial F1FO-ATP synthase elements opened the door for a renaissance of structure-based target identification and inhibitor design. In this study, we present the GaMF1.39 antimycobacterial compound, targeting the rotary subunit γ of the biological engine. The compound is bactericidal, inhibits infection ex vivo, and displays enhanced anti-tuberculosis activity in combination with ETC inhibitors, which promises new strategies to shorten tuberculosis chemotherapy.

Solution structure of FK506-binding protein 12 from Aedes aegypti.

Dengue remains one of the major public concerns as the virus eludes the immune response. Currently, no vaccines or antiviral therapeutics are available for dengue prevention or treatment. Immunosuppressive drug FK506 shows an antimalarial activity, and its molecular target, FK506-binding protein (FKBP), was identified in human Plasmodium parasites. Likewise, a conserved FKBP family protein has also been identified in Aedes aegypti (AaFKBP12), which is expected to play a similar role in the life cycle of Aedes aegypti, the primary vector of dengue virus infection. As FKBPs belong to a highly conserved class of immunophilin family and are involved in key biological regulations, they are considered as attractive pharmacological targets. In this study, we have determined the nuclear magnetic resonance solution structure of AaFKBP12, a novel FKBP member from Aedes aegypti, and presented its structural features, which may facilitate the design of potential inhibitory ligands against the dengue-transmitting mosquitoes.

Atomic solution structure of Mycobacterium abscessus F-ATP synthase subunit ε and identification of Ep1MabF1 as a targeted inhibitor.

Mycobacterium abscessus (Mab) is a nontuberculous mycobacterium of increasing clinical relevance. The rapidly growing opportunistic pathogen is intrinsically multi-drug-resistant and causes difficult-to-cure lung disease. Adenosine triphosphate, generated by the essential F1 FO ATP synthase, is the major energy currency of the pathogen, bringing this enzyme complex into focus for the discovery of novel antimycobacterial compounds. Coupling of proton translocation through the membrane-embedded FO sector and ATP formation in the F1 headpiece of the bipartite F1 FO ATP synthase occurs via the central stalk subunits γ and ε. Here, we used solution NMR spectroscopy to resolve the first atomic structure of the Mab subunit ε (Mabε), showing that it consists of an N-terminal ß-barrel domain (NTD) and a helix-loop-helix motif in its C-terminal domain (CTD). NMR relaxation measurements of Mabε shed light on dynamic epitopes and amino acids relevant for coupling processes within the protein. We describe structural differences between other mycobacterial ε subunits and Mabε's lack of ATP binding. Based on the structural insights, we conducted an in silico inhibitor screen. One hit, Ep1MabF1, was shown to inhibit the growth of Mab and bacterial ATP synthesis. NMR titration experiments and docking studies described the binding epitopes of Ep1MabF1 on Mabε. Together, our data demonstrate the potential to develop inhibitors targeting the ε subunit of Mab F1 FO ATP synthase to interrupt the coupling process.

Mutational Analysis of Mycobacterial F-ATP Synthase Subunit δ Leads to a Potent δ Enzyme Inhibitor.

While many bacteria are able to bypass the requirement for oxidative phosphorylation when grown on carbohydrates, Mycobacterium tuberculosis is unable to do so. Differences of amino acid composition and structural features of the mycobacterial F-ATP synthase (α3:ß3:γ:δ:ε:a:b:b':c9) compared to its prokaryotic or human counterparts were recently elucidated and paved avenues for the discovery of molecules interfering with various regulative mechanisms of this essential energy converter. In this context, the mycobacterial peripheral stalk subunit δ came into focus, which displays a unique N-terminal 111-amino acid extension. Here, mutants of recombinant mycobacterial subunit δ were characterized, revealing significant reduction in ATP synthesis and demonstrating essentiality of this subunit for effective catalysis. These results provided the basis for the generation of a four-feature model forming a δ receptor-based pharmacophore and to identify a potent subunit δ inhibitor DeMF1 via in silico screening. The successful targeting of the δ subunit demonstrates the potential to advance δ's flexible coupling as a new area for the development of F-ATP synthase inhibitors.

Structural and Mechanistic Insights into Mycobacterium abscessus Aspartate Decarboxylase PanD and a Pyrazinoic Acid-Derived Inhibitor.

Mycobacterium tuberculosis (Mtb) aspartate decarboxylase PanD is required for biosynthesis of the essential cofactor coenzyme A and targeted by the first line drug pyrazinamide (PZA). PZA is a prodrug that is converted by a bacterial amidase into its bioactive form pyrazinoic acid (POA). Employing structure-function analyses we previously identified POA-based inhibitors of Mtb PanD showing much improved inhibitory activity against the enzyme. Here, we performed the first structure-function studies on PanD encoded by the nontuberculous mycobacterial lung pathogen Mycobacterium abscessus (Mab), shedding light on the differences and similarities of Mab and Mtb PanD. Solution X-ray scattering data provided the solution structure of the entire tetrameric Mab PanD, which in comparison to the structure of the derived C-terminal truncated Mab PanD1-114 mutant revealed the orientation of the four flexible C-termini relative to the catalytic core. Enzymatic studies of Mab PanD1-114 explored the essentiality of the C-terminus for catalysis. A library of recombinant Mab PanD mutants based on structural information and PZA/POA resistant PanD mutations in Mtb illuminated critical residues involved in the substrate tunnel and enzymatic activity. Using our library of POA analogues, we identified (3-(1-naphthamido)pyrazine-2-carboxylic acid) (analogue 2) as the first potent inhibitor of Mab PanD. The inhibitor shows mainly electrostatic- and hydrogen bonding interaction with the target enzyme as explored by isothermal titration calorimetry and confirmed by docking studies. The observed unfavorable entropy indicates that significant conformational changes are involved in the binding process of analogue 2 to Mab PanD. In contrast to PZA and POA, which are whole-cell inactive, analogue 2 exerts appreciable antibacterial activity against the three subspecies of Mab.

Targeting C-terminal Helical bundle of NCOVID19 Envelope (E) protein.

One of the most crucial characteristic traits of Envelope (E) proteins in the severe acute respiratory syndrome SARS-CoV-1 and NCOVID19 viruses is their membrane-associated oligomerization led ion channel activity, virion assembly, and replication. NMR spectroscopic structural studies of envelope proteins from both the SARS CoV-1/2 reveal that this protein assembles into a homopentamer. Proof of concept studies via truncation mutants on either transmembrane (VFLLV), glycosylation motif (CACCN), hydrophobic helical bundle (PVYVY) as well as replacing C-terminal "DLLV" segments or point mutants such as S68, E69 residues with cysteine have significantly reduced viral titers of SARS-CoV-1. In this present study, we have first developed SARS-2 E protein homology model based on the pentamer coordinates of SARS-CoV-1 E protein (86.4% structural identity) with good stereochemical quality. Next, we focused on the glycosylation motif and hydrophobic helical bundle regions of E protein shown to be important for viral replication. A four feature (4F) model comprising of an acceptor targeting S60 hydroxyl group, a donor feature anchoring the C40 residue, and two hydrophobic features anchoring the V47 L28, L31, Y55, and P51 residues formed the protein based pharmacophore model targeting the glycosylation motif and helical bundle of E protein. Database screening with this 4F protein pharmacophore, ADMET property filtering on enamine small molecule discovery collection yielded a focused library of ~7000 hits. Further molecular docking and visual inspection of docked pose interactions at the above mention V47 L28, L31, Y55, P51, S60, C40 residues led to the identification of 10 best hits. Our STD NMR binding assay results demonstrate that the ligand 3, 2-(2-amino-2-oxo-ethoxy)-N-benzyl-benzamide, binds to NCOVID19 E protein with a binding affinity (KD) of 141.7 ± 13.6 µM. Furthermore, the ligand 3 also showed binding to C-terminal peptide (NR25) as evidenced with the STD spectrums of wild type E protein would serve to confirm the involvement of C-terminal helical bundle as envisaged in this study.

Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ.

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:ß3:γ:δ:ε:a:b:b':c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

Deciphering the Role of Ion Channels in Early Defense Signaling against Herbivorous Insects.

Plants and insect herbivores are in a relentless battle to outwit each other. Plants have evolved various strategies to detect herbivores and mount an effective defense system against them. These defenses include physical and structural barriers such as spines, trichomes, cuticle, or chemical compounds, including secondary metabolites such as phenolics and terpenes. Plants perceive herbivory by both mechanical and chemical means. Mechanical sensing can occur through the perception of insect biting, piercing, or chewing, while chemical signaling occurs through the perception of various herbivore-derived compounds such as oral secretions (OS) or regurgitant, insect excreta (frass), or oviposition fluids. Interestingly, ion channels or transporters are the first responders for the perception of these mechanical and chemical cues. These transmembrane pore proteins can play an important role in plant defense through the induction of early signaling components such as plasma transmembrane potential (Vm) fluctuation, intracellular calcium (Ca2+), and reactive oxygen species (ROS) generation, followed by defense gene expression, and, ultimately, plant defense responses. In recent years, studies on early plant defense signaling in response to herbivory have been gaining momentum with the application of genetically encoded GFP-based sensors for real-time monitoring of early signaling events and genetic tools to manipulate ion channels involved in plant-herbivore interactions. In this review, we provide an update on recent developments and advances on early signaling events in plant-herbivore interactions, with an emphasis on the role of ion channels in early plant defense signaling.

Antiviral activity against Middle East Respiratory Syndrome coronavirus by Montelukast, an anti-asthma drug.

Middle East Respiratory Syndrome (MERS) is a respiratory disease caused by a coronavirus (MERS-CoV). Since its emergence in 2012, nosocomial amplifications have led to its high epidemic potential and mortality rate of 34.5%. To date, there is an unmet need for vaccines and specific therapeutics for this disease. Available treatments are either supportive medications in use for other diseases or those lacking specificity requiring higher doses. The viral infection mode is initiated by the attachment of the viral spike glycoprotein to the human Dipeptidyl Peptidase IV (DPP4). Our attempts to screen antivirals against MERS led us to identify montelukast sodium hydrate (MSH), an FDA-approved anti-asthma drug, as an agent attenuating MERS-CoV infection. We showed that MSH directly binds to MERS-CoV-Receptor-Binding Domain (RBD) and inhibits its molecular interaction with DPP4 in a dose-dependent manner. Our cell-based inhibition assays using MERS pseudovirions demonstrated that viral infection was significantly inhibited by MSH and was further validated using infectious MERS-CoV culture. Thus, we propose MSH as a potential candidate for therapeutic developments against MERS-CoV infections.