Fungal toxin fusicoccin enhances plant growth by upregulating 14-3-3 interaction with plasma membrane H-ATPase.

Fusicoccin-A (FC-A) is a diterpene glucoside produced by a pathogenic fungus. Since its discovery, FC-A has been widely recognized as a phytotoxin that induces stomatal opening and leaf wilting, eventually leading to plant death. In this study, we present the first evidence that FC-A enhances plant growth by stabilizing the protein-protein interaction between plasma membrane (PM) H-ATPase and 14-3-3 in guard cells.

Functional evaluation of an electrophilic focused library to identify a covalent inhibitor against intrinsically disordered circadian clock transcription factors.

In vitro screening of a focused library of compounds containing an electrophilic warhead identified N-chloroacetyl-bis(trifluoromethyl)aniline derivative 15 as a potent inhibitor of BMAL1-CLOCK heterodimer binding to an E-box DNA fragment. Kinetic analysis of thiol-reactivity demonstrated that iodoacetamide and structurally related 20 are significantly more reactive than or equally reactive as 15, respectively, whereas none inhibited BMAL1-CLOCK interaction with the E-box DNA fragment. These results suggest that 15 binds and reacts with a specific nucleophilic residue.

Isoform-Selective Fluorescent Labeling of 14-3-3σ by Acrylamide-Containing Fusicoccins.

The 14-3-3 family of proteins is central to the regulation of signaling pathways driven by serine/threonine kinases. In humans, 14-3-3 consists of seven highly conserved isoforms, yet the function of each isoform remains to be fully elucidated. Synthetic agents capable of isoform-specific fluorescent labeling of 14-3-3 would provide a useful tool for studying in depth the biological roles of isoforms.

Structure-activity-relationship study of semi-synthetically modified fusicoccins on their stabilization effect for 14-3-3-phospholigand interactions.

The diterpene glucoside fusicoccin-A (FC-A) is a fungal phytotoxin that stabilizes the interaction of plant 14-3-3 protein and plasma membrane H-ATPase by forming a stable ternary complex. Previous studies demonstrated that structurally modified FC-A derivatives exhibit significant antitumor activities but their synthesis involves an explosive reagent, limiting their utility and opportunities for further structure-activity-relationship studies. In this study, we synthesized a series of FC derivatives by introducing various substituents on the fusicoccan scaffold and on the glucoside moiety, and evaluated their stabilization effects on the binding of 14-3-3 to fluorescently labeled mode-1 and mode-3 phosphopeptides.

Expression and purification of DYRK1A kinase domain in complex with its folding intermediate-selective inhibitor FINDY.

The kinase DYRK1A phosphorylates substrate proteins that are involved in the progression of many diseases. DYRK1A also phosphorylates its own residues on key elements intramolecularly to activate and stabilize itself during the folding process. Once the folding process of DYRK1A has completed, it can no longer catalyzes the intramolecular reaction, suggesting that a transitional intermediate state that catalyzes the autophosphorylation exists.

Design, synthesis, and functional evaluation of triazine-based bivalent agents that simultaneously target the active site and hot spot of phosphatase Cdc25B.

Cdc25B phosphatase catalyzes the dephosphorylation and activation of cyclin-dependent kinases 2 (CDK2/CycA) and their overexpression has been reported in cancers. Although Cdc25B has received much attention as a drug target, its flat and featureless surface makes it challenging to develop new agents targeting this protein. In this study, we investigated the rational design of a series of bivalent triazine-based derivatives with the aim of simultaneously targeting the active site and the remote hotspot critical for the interaction with CDK2/CycA.

Intrinsically Disordered Proteins as Regulators of Transient Biological Processes and as Untapped Drug Targets.

Intrinsically disordered proteins (IDPs) are critical players in the dynamic control of diverse cellular processes, and provide potential new drug targets because their dysregulation is closely related to many diseases. This review focuses on several medicinal studies that have identified low-molecular-weight inhibitors of IDPs. In addition, clinically relevant liquid-liquid phase separations-which critically involve both intermolecular interactions between IDPs and their posttranslational modification-are analyzed to understand the potential of IDPs as new drug targets.

Identification of synthetic inhibitors for the DNA binding of intrinsically disordered circadian clock transcription factors.

Essential components of the human circadian clock, BMAL1 and CLOCK, which are intrinsically disordered transcription factors, were expressed and subjected to a fluorescent in vitro binding assay using an E-box DNA fragment. Screening of a chemical library identified 5,8-quinoxalinedione (1), which was found to inhibit binding of the heterodimer BMAL1/CLOCK to E-box at low micromolar concentrations.

Copper-Free Huisgen Cycloaddition for the 14-3-3-Templated Synthesis of Fusicoccin-Peptide Conjugates.

Mid-sized molecules have emerged as an attractive chemical space and potentially provide a robust basis for the development of synthetic agents to control intracellular protein interactions. However, the limited cell permeability and chemical tractability of such agents remain to be addressed. We envisioned that target-templated synthesis of such mid-sized molecules might provide a solution.

A Guanidyl-Based Bivalent Peptidomimetic Inhibits K-Ras Prenylation and Association with c-Raf.

Unusual lipid modification of K-Ras makes Ras-directed cancer therapy a challenging task. Aiming to disrupt electrostatic-driven protein-protein interactions (PPIs) of K-Ras with FTase and GGTase I, a series of bivalent dual inhibitors that recognize the active pocket and the common acidic surface of FTase and GGTase I were designed. The structure-activity-relationship study resulted in 8 b, in which a biphenyl-based peptidomimetic FTI-277 was attached to a guanidyl-containing gallate moiety through an alkyl linker.

Dipicolylamine/Metal Complexes that Promote Direct Cell-Membrane Penetration of Octaarginine.

Marked promotion of membrane permeation of a cell-penetrating peptide, octaarginine (R8), was attained by attachment to a single 2,2'-dipicolylamine moiety (DPA-R8) that forms 1:1 complexes with metal ions. Studies using giant unilamellar vesicles demonstrated that DPA targets phospholipids and enhances R8 binding to the membranes in the presence of metal ions. While DPA/Zn(II) complex has been most frequently employed for chelate formation with phosphates, Ni(II) had the most prominent effect on the membrane binding and penetration of DPA-R8.

Structural Effects of Fusicoccin upon Upregulation of 14-3-3-Phospholigand Interaction and Cytotoxic Activity.

Fusicoccins (FCs) exhibit various cellular activities in mammalian cells, but details of the mechanism of action are not fully understood. In this study, we synthesized two pairs of model derivatives of FCs differing only in the presence and absence of a 12-hydroxyl group and evaluated their binding to a 14-3-3 protein together with various mode 1 and mode 3 phosphopeptide ligands. Our results demonstrate that the 12-hydroxyl group hampers binding to 14-3-3 with mode 1 phospholigands, presumably due to steric repulsion with the i+2 residue.

Intracellular Generation of a Diterpene-Peptide Conjugate that Inhibits 14-3-3-Mediated Interactions.

Synthetic agents that disrupt intracellular protein-protein interactions (PPIs) are highly desirable for elucidating signaling networks and developing new therapeutics. However, designing cell-penetrating large molecules equipped with the many functional groups necessary for binding to large interfaces remains challenging. Here, we describe a rational strategy for the intracellular oxime ligation-mediated generation of an amphipathic bivalent inhibitor composed of a peptide and diterpene natural product, fusicoccin, which binds 14-3-3 protein with submicromolar affinity.

RNA-directed amino acid coupling as a model reaction for primitive coded translation.

The stereochemical theory claims that primitive coded translation initially occurred in the RNA world by RNA-directed amino acid coupling. In this study, we show that the HIV Tat aptamer RNA is capable of recognizing two consecutive arginine residues within the Tat peptide, thus demonstrating how RNA might be able to position two amino acids for sequence-specific coupling. We also show that this RNA can act as a template to accelerate the coupling of a single arginine residue to the N-terminal arginine residue of a peptide primer.

Module assembly for designing multivalent mid-sized inhibitors of protein-protein interactions.

Developing clinically relevant synthetic agents that are capable of disrupting protein-protein interactions (PPIs) is now a major goal of scientific research. In an effort to explore new methodologies that are applicable to the design of synthetic PPI inhibitors, we examined a strategy based on the assembly of small module compounds to create multivalent mid-sized agents. This personal account describes three particular approaches based on module assembly: metal-chelating-based ligand assembly, covalent chemical ligation templated by a targeted protein, and bivalent inhibitor design for simultaneous targeting of the active pocket and protein surface.

Stabilization of physical RAF/14-3-3 interaction by cotylenin A as treatment strategy for RAS mutant cancers.

One-third of all human cancers harbor somatic RAS mutations. This leads to aberrant activation of downstream signaling pathways involving the RAF kinases. Current ATP-competitive RAF inhibitors are active in cancers with somatic RAF mutations, such as BRAF(V600) mutant melanomas.

A semisynthetic fusicoccane stabilizes a protein-protein interaction and enhances the expression of K+ channels at the cell surface.

Small-molecule stabilization of protein-protein interactions is an emerging field in chemical biology. We show how fusicoccanes, originally identified as fungal toxins acting on plants, promote the interaction of 14-3-3 proteins with the human potassium channel TASK-3 and present a semisynthetic fusicoccane derivative (FC-THF) that targets the 14-3-3 recognition motif (mode 3) in TASK-3. In the presence of FC-THF, the binding of 14-3-3 proteins to TASK-3 was increased 19-fold and protein crystallography provided the atomic details of the effects of FC-THF on this interaction.

Antimicrobial N-(2-chlorobenzyl)-substituted hydroxamate is an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase.

N-(2-Chlorobenzyl)-substituted hydroxamate, readily produced by hydrolysis of ketoclomazone, was identified as an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), with an IC50 value of 1.0 μM. The compound inhibited the growth of Haemophilus influenzae.

Chemical ligation of epoxide-containing fusicoccins and peptide fragments guided by 14-3-3 protein.

The template effect of 14-3-3 protein on the chemical ligation of fusicoccin derivatives containing an epoxide group and the pentapeptide QSYDC was investigated. HPLC analysis of the epoxide-opening reaction demonstrated that 14-3-3ζ protein improves the yield of the conjugate product compared to the protein-free control.

Peptidomimetic modification improves cell permeation of bivalent farnesyltransferase inhibitors.

Bivalent enzyme inhibitors, in which a surface binding module is linked to an active site binding module through a spacer, are a robust approach for site-selectively delivering a minimally-sized agent to a protein surface to regulate its functions, such as protein-protein interactions (PPIs). Previous research revealed that these agents effectively disrupt the interaction between farnesyltransferase (FTase) and the C-terminal region of K-Ras4B protein. However, the whole cell activity of these peptide-based agents is limited due to their low membrane permeability.

Protein recognition of hetero-/homoleptic ruthenium(II) tris(bipyridine)s for α-chymotrypsin and cytochrome c.

We examined the relationship between the structures of hetero-/homoleptic ruthenium(II) tris(bipyridine) metal complexes (Ru(II)(bpy)(3)) and their binding properties for α-chymotrypsin (ChT) and cytochrome c (cyt c). Heteroleptic compound 1a binds to both ChT and cyt c in 1:1 ratio, whereas homoleptic 2 forms 1:2 protein complex with ChT but 1:1 complex with cyt c. These results suggest that the structure of the recognition cavity in Ru(II)(bpy)(3) can be designed for shape complementarity to the targeted proteins.

A novel fusicoccin derivative preferentially targets hypoxic tumor cells and inhibits tumor growth in xenografts.

Malignant cells in solid tumors survive under prolonged hypoxia and can be a source of resistance to current cancer therapies. Tumor hypoxia is also associated with a more malignant phenotype and poor survival in cancer patients. Recent progress in our understanding of the biology of tumor cells under hypoxia has led to increased attention on targeting hypoxia for cancer therapy.

Phosphopeptide-dependent labeling of 14-3-3 ζ proteins by fusicoccin-based fluorescent probes.

Fluorescent combination: Cell-penetrating probes derived from the diterpene fusicoccin can form ternary complexes with 14-3-3 proteins and phosphopeptide ligands, whereupon the probes site-specifically attach a fluorescent tag onto the surface of the 14-3-3 proteins.

Bivalent inhibitors for disrupting protein surface-substrate interactions and for dual inhibition of protein prenyltransferases.

Low-molecular-weight compounds that disrupt protein−protein interactions (PPIs) have tremendous potential applications as clinical agents and as chemical probes for investigating intracellular PPI networks. However, disrupting PPIs is extremely difficult due to the large, flat interfaces of many proteins, which often lack structurally defined cavities to which drug-like molecules could bind in a thermodynamically favorable manner. Here, we describe a series of bivalent compounds that anchor to the enzyme active site to deliver a minimally sized surface-binding module to the targeted surface involved in transient PPI with a substrate.

Protein surface recognition by dendritic ruthenium(II) tris(bipyridine) complexes.

We report protein surface binding of dendritic ruthenium(ii) tris(bipyiridine) complexes to alpha-chymotrypsin, resulting in a 1 : 1 and 1 : 2 protein complex formation as well as inhibition of the enzyme activity.