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. This low-molecular-weight compound may serve as a useful lead for further development of BMAL1-CLOCK inhibitors.

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. In this study, the 14-3-3σ isoform was evaluated, which possesses a unique Cys38, and a natural product-based fluorescent labeling agent was designed by introducing an acrylamide group and a fluorescent dye to fusicoccin (FC). In vitro evaluation demonstrated that 12-hydroxy 1 and 2 exhibit 14-3-3σ selective labeling activity over 14-3-3ζ in the presence of a mode-3 phospholigand. Furthermore, 2 was shown to label 14-3-3σ in cell lysate in the presence of a C-terminal mode-3 phosphopeptide derived from ERα, with no apparent nonspecific labeling. These results indicate that 2 is capable of selective fluorescent detection of 14-3-3σ upon binding to mode-3 phospholigand under biologically relevant conditions.

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. In the previous study, we identified a small molecule, designated as FINDY, that selectively inhibits the folding intermediate of DYRK1A. Although evidence has suggested that FINDY targets the ATP-binding pocket of DYRK1A, it remains elusive as to whether the DYRK1A kinase domain could be purified as a complex with FINDY. In this study, we successfully expressed and purified the kinase domain of DYRK1A in complex with FINDY. The DYRK1A kinase domain was expressed as a fusion protein with a hexahistidine tag and ZZ-domain (His-ZZ-DYRK1A) at 6 °C by using a cold shock induction system in Escherichia coli cells. The cells were incubated with FINDY. The cell pellets were gently extracted on ice and subjected to immobilized-metal affinity chromatography. The amount of FINDY in the elution fraction was measured by UV absorbance specific for FINDY. The eluate contained FINDY with the ratio of FINDY to DYRK1A protein being 0.15 in quadruplicate experiments. Thus, this study demonstrates the direct interaction between the DYRK1A kinase domain and FINDY, paving the way for structural determination of the complex.

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. The results showed that introducing an amino group at the 6'-position of the glucoside moiety improves stabilization. Furthermore, cell-based evaluation demonstrated that 6'-amino benzyl 21b exhibits higher antiproliferative activity than previously developed FC agents.

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. Compounds 1e and 10, containing aromatic residues, were shown to inhibit Cdc25B activity selectively over Cdc25A at low micromolar concentration.

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.

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. Facile cytosolic distribution of DPA-R8 was also attained in a few minutes in the presence of Ni(II). Analysis of the cellular uptake methods of DPA-R8/Ni(II) suggested the involvement of direct permeation through cell membrane without the use of endocytosis. The applicability of this system to the intracellular delivery of bioactive compounds was exemplified using a peptidomimetic farnesyltransferase inhibitor, FTI277.

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. Cell-based evaluation demonstrated that 8 b exhibited substantial inhibition of K-Ras processing without apparent interference with Rap-1A processing. Fluorescent imaging showed that 8 b disrupts localization of K-Ras to the plasma membrane and impairs interaction with c-Raf, whereas only FTI-277 was found to be inactive. These results suggest that targeting the PPI interface of K-Ras may provide an alternative method of inhibiting K-Ras.

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. Furthermore, cell-based evaluations showed that only non-substituted FCs exhibit significant cytotoxicity and all 12-hydroxyl derivatives were inactive, demonstrating a clear correlation with their ability to form ternary complexes with 14-3-3 and a mode 1 ligand. These results suggest that binding to 14-3-3 and a partner protein(s) possessing a mode 1 sequence plays a role in the mechanism of action of 12-non-substituted FCs.

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. Our results demonstrate that co-treatment of cells with small module molecules, the aldehyde-containing fusicoccin 1 and the aminooxy-containing peptide 2, generates the corresponding conjugate 3 in cells, resulting in significant cytotoxicity. In contrast, chemically synthesized 3 is not cytotoxic, likely due to its inability to penetrate cells. Compound 3, but not 1 or 2, disrupts endogenous 14-3-3/cRaf interactions, suggesting that cell death is caused by inhibition of 14-3-3 activity. These results suggest that intracellular generation of large-sized molecules may serve as a new approach for modulating PPIs.

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. The results might have implications for our understanding of the origin of translation.

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. These strategies were shown to be useful for synthesizing minimally sized synthetic agents for targeting PPIs and may enable development of agents that are applicable to inhibition of intracellular PPIs.

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. In this study, we tested a peptidomimetic modification of these bivalent agents using a previously developed inhibitor, FTI-249, and evaluated their cell permeability and biological activity in cells. Confocal cell imaging using fluorescently-labeled agents showed that the peptidomimetic 3-BODIPY penetrated cells, while the peptide-based 1-BODIPY did not. Cell-based evaluation demonstrated that peptidomimetic 3 at a concentration of 100µM inhibited HDJ-2 processing in cells, indicating that this peptidomimetic modification improves cell permeability, thus leading to enhanced whole cell activity of the bivalent compounds.

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. In addition, Ru(II)(bpy)(3) complexes were found to be potent inhibitors of cyt c reduction and to permeate A549 cells.

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. These compounds are capable of significantly inhibiting enzymatic reactions involving protein surface−substrate interaction in the single-digit nanomole range. Inhibitors of farnesyltransferase (FTase), which possesses a negatively charged local area on its α-subunit, were designed by attaching a module derived from a branched monoamine-containing gallate to a conventional active-site-directed CVIM tetrapeptide using an alkyl spacer. A significant improvement in inhibitory activity (>200-fold) against farnesylation of the K-Ras4B peptide was observed when the gallate module was attached to the CVIM tetrapeptide. Furthermore, the bivalent compounds had submicromolar inhibitory activity against geranylgeranylation of the K-Ras4B peptide catalyzed by GGTase I, which has an α-subunit identical to that of FTase. The anchoring strategy we describe would be useful for designing a new class of PPI inhibitors as well as dual enzyme inhibitors targeting common surface structures.

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. Here, we exploited a copper-free Huisgen cycloaddition for template synthesis using a peptide fragment containing a 4,8-diazacyclononyne (DACN) moiety and an azide-containing fusicoccin derivative in the presence or absence of recombinant 14-3-3ζ protein in vitro. Time-course changes in the yield of products demonstrated that the reaction was accelerated in the presence of 14-3-3 and one of the regioisomers was generated predominantly, supporting the template effect.

In vivo antiviral efficacy of prenylation inhibitors against hepatitis delta virus.

Hepatitis delta virus (HDV) can dramatically worsen liver disease in patients coinfected with hepatitis B virus (HBV). No effective medical therapy exists for HDV. The HDV envelope requires HBV surface antigen proteins provided by HBV. Once inside a cell, however, HDV can replicate its genome in the absence of any HBV gene products. In vitro, HDV virion assembly is critically dependent on prenyl lipid modification, or prenylation, of its nucleocapsid-like protein large delta antigen. To overcome limitations of current animal models and to test the hypothesis that pharmacologic prenylation inhibition can prevent the production of HDV virions in vivo, we established a convenient mouse-based model of HDV infection capable of yielding viremia. Such mice were then treated with the prenylation inhibitors FTI-277 and FTI-2153. Both agents were highly effective at clearing HDV viremia. As expected, HDV inhibition exhibited duration-of-treatment dependence. These results provide the first preclinical data supporting the in vivo efficacy of prenylation inhibition as a novel antiviral therapy with potential application to HDV and a wide variety of other viruses.

Geranylgeranyltransferase I inhibitor GGTI-2154 induces breast carcinoma apoptosis and tumor regression in H-Ras transgenic mice.

Treatment of H-Ras transgenic mice with the geranylgeranyltransferase I (GGTase I) inhibitor GGTI-2154 results not only in halting the growth of aggressive breast tumors but actually in inducing the regression (54 +/- 3%) of all 19 tumors analyzed. The farnesyltransferase (FTase) inhibitor FTI-2148 induced an average of 87 +/- 3% regression in the 13 tumors analyzed. GGTase I, but not FTase, is inhibited in breast tumors after treatment with GGTI-2154, whereas in tumors from mice treated with FTI-2148, only FTase is inhibited. The processing of the geranylgeranylated proteins RhoA, Rap1, and R-Ras, but not the farnesylated proteins H-Ras and HDJ-2, is inhibited in tumors obtained from mice treated with GGTI-2154. GGTI-2154 and FTI-2148 suppress constitutively activated phospho-Erk1/2 and phospho-Akt, induce apoptosis, and induce differentiation toward ductolobular breast epithelium. The data demonstrate that geranylgeranylated proteins are critical in H-Ras oncogenesis in vivo and give strong support for GGTase I as a target for anticancer drug discovery.