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

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:β3: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

Revisiting De Novo drug design : receptor based pharmacophore screening

De novo drug design methods such as receptor or protein based pharmacophore modeling present a unique opportunity to generate novel ligands by employing the potential binding sites even when no explicit ligand information is known for a particular target. Recent developments in molecular modeling programs have enhanced the ability of early programs such as LUDI or Pocket that not only identify the key interactions or hot spots at the suspected binding site, but also and convert these hot spots into three-dimensional search queries and virtual screening of the property filtered synthetic libraries. Together with molecular docking studies and consensus scoring schemes they would enrich the lead identification processes. In this review, we discuss the ligand and receptor based de novo drug design approaches with selected examples.Accepted versio

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

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.National Research Foundation (NRF)Submitted/Accepted versionThe authors are supported by the National Research Foundation (NRF) Singapore, through the NRF Competitive Research Programme (CRP), Grant Award Number NRF-CRP27-2021-0002

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 “super-complex” since it combines three cytochrome oxidases—cytochrome bc,cytochromec, 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 charac- terization of this supercomplex, paving the way for new inhibitor targets and molecules. Here,weproducedandpurified 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, thecounterpart 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 mycobac- terium-specific a3-helix of cytochrome cI and interferes with oxygen consumption by interrupting electron translocation via the cIcII head. The successful identification of anew cyt-bcc:aa3 inhibitor demonstrates the potential of a structure-mechanism-based approach for novel compound development.National Research Foundation (NRF)Submitted/Accepted versionThis study was supported by National Research Foundation (NRF) Singapore, NRF Competitive Research Program (CRP), grants NRF-CRP18-2017-01 and NRF-CRP27-2021-0002. V.M. acknowledges a NTU Research Scholarship. C.-F.W.’s Ph.D. scholarship was funded by an NRF CRP grant (award NRF-CRP18-2017-01)

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

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:β3: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.National Research Foundation (NRF)Submitted/Accepted versionThis research was supported by the National Research Foundation (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF-CRP18-2017- 01), and the Deutsche Forschungsgemeinschaft via SFB807

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 :β3 :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.National Research Foundation (NRF)Published versionThis research was supported by the National Research Foundation (NRF) Singapore, Competitive Research Programme (CRP), Grant Award Number NRF-CRP18-2017-01, and the Deutsche Forschungsgemeinschaft via SFB807

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 β-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.National Research Foundation (NRF)Submitted/Accepted versionThis research was supported by the National Research Foundation (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF–CRP18–2017–01, including the PhD scholarship of C-FW