Synthesis and Characterization of Sulfonamide-Containing Naphthalimides as Fluorescent Probes.

A tumor-targeting fluorescent probe has attracted increasing interest in fluorescent imaging for the noninvasive detection of cancers in recent years. Sulfonamide-containing naphthalimide derivatives (SN-2NI, SD-NI) were synthesized by the incorporation of N-butyl-4-ethyldiamino-1,8-naphthalene imide (NI) into sulfonamide (SN) and sulfadiazine (SD) as the tumor-targeting groups, respectively. These derivatives were further characterized by mass spectrometry (MS), nuclear magnetic resonance spectroscopy (1H NMR), Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV), and a fluorescence assay. In vitro properties, including cell cytotoxicity and the cell uptake of tumor cells, were also evaluated. Sulfonamide-containing naphthalimide derivatives possessed low cell cytotoxicity to B16F10 melanoma cells. Moreover, SN-2NI and SD-NI can be taken up highly by B16F10 cells and then achieve good green fluorescent images in B16F10 cells. Therefore, sulfonamide-containing naphthalimide derivatives can be considered to be the potential probes used to target fluorescent imaging in tumors.

Discovery of novel 1,8-naphthalimide piperazinamide based benzenesulfonamides derivatives as potent carbonic anhydrase IX inhibitors and ferroptosis inducers for the treatment of triple-negative breast cancer.

A novel series of 1,8-naphthalimide piperazinamide based benzenesulfonamides derivatives were designed and synthesized as carbonic anhydrase IX (CA IX) inhibitors and ferroptosis inducers for the treatment of triple-negative breast cancer (TNBC). The representative compound 9o exhibited more potent inhibitory activity and selective against CA IX over off-target CA II, compared with positive control SLC-0111. Molecular docking study was also performed to gain insights into the binding interactions of 9o in the binding pocket of CAIX. Moreover, compound 9o exhibited superior antitumor activities against breast cancer cells under hypoxia than that of normoxia conditions. Mechanism studies revealed that compound 9o could act as DNA intercalator and effectively suppressed cell migration, arrested the cell cycle at G1/S phase and induced apoptosis in MDA-MB-231 cells, while inducing ferroptosis accompanied by the dissipation of MMP and the elevation intracellular levels of ROS. Notably, in vivo studies demonstrated that 9o effectively inhibited tumor growth and metastasis in a highly metastatic murine breast cancer 4 T1 xenograft model. Taken together, this study suggests that compound 9o represents a potent and selective CA IX inhibitor and ferroptosis inducer for the treatment of TNBC.

Design, synthesis and structure-activity relationship of 1,8-naphthalimide derivatives as highly potent hCYP1B1 inhibitors.

Human cytochrome P450 1B1 enzyme (hCYP1B1), a member of hCYP1 subfamily, plays a crucial role in multiple diseases by participating in many metabolic pathways. Although a suite of potent hCYP1B1 inhibitors have been previously reported, most of them also act as aryl hydrocarbon receptor (AhR) agonists that can up-regulate the expression of hCYP1B1 and then counteract their inhibitory potential in living systems. This study aimed to develop novel efficacious hCYP1B1 inhibitors that worked well in living cells but without AhR agonist effects. For these purposes, a series of 1,8-naphthalimide derivatives were designed and synthesized, and their structure-activity relationships (SAR) as hCYP1B1 inhibitors were analyzed. Following three rounds SAR studies, several potent hCYP1B1 inhibitors were discovered, among which compound 3n was selected for further investigations owing to its extremely potent anti-hCYP1B1 activity (IC50 = 0.040 nM) and its blocking AhR transcription activity in living cells. Inhibition kinetic analyses showed that 3n potently inhibited hCYP1B1 via a mix inhibition manner, showing a Ki value of 21.71 pM. Docking simulations suggested that introducing a pyrimidine moiety to the hit compound (1d) facilitated 3n to form two strong interactions with hCYP1B1/heme, viz., the C-Br⋯π halogen bond and the N-Fe coordination bond. Further investigations demonstrated that 3n (5 µM) could significantly reverse the paclitaxel (PTX) resistance in H460/PTX cells, evidenced by the dramatically reduced IC50 values, from 632.6 nM (PTX alone) to 100.8 nM (PTX plus 3n). Collectively, this study devised a highly potent hCYP1B1 inhibitor (3n) without AhR agonist effect, which offered a promising drug candidate for overcoming hCYP1B1-associated drug resistance.

Identification of naphthalimide-derivatives as novel PBD-targeted polo-like kinase 1 inhibitors with efficacy in drug-resistant lung cancer cells.

Targeting polo-box domain (PBD) small molecule for polo-like kinase 1 (PLK1) inhibition is a viable alternative to target kinase domain (KD), which could avoid pan-selectivity and dose-limiting toxicity of ATP-competitive inhibitors. However, their efficacy in these settings is still low and inaccessible to clinical requirement. Herein, we utilized a structure-based high-throughput virtual screen to find novel chemical scaffold capable of inhibiting PLK1 via targeting PBD and identified an initial hit molecule compound 1a. Based on the lead compound 1a, a structural optimization approach was carried out and several series of derivatives with naphthalimide structural motif were synthesized. Compound 4Bb was identified as a new potent PLK1 inhibitor with a KD value of 0.29 µM. 4Bb could target PLK1 PBD to inhibit PLK1 activity and subsequently suppress the interaction of PLK1 with protein regulator of cytokinesis 1 (PRC1), finally leading to mitotic catastrophe in drug-resistant lung cancer cells. Furthermore, 4Bb could undergo nucleophilic substitution with the thiol group of glutathione (GSH) to disturb the redox homeostasis through exhausting GSH. By regulating cell cycle machinery and increasing cellular oxidative stress, 4Bb exhibited potent cytotoxicity to multiple cancer cells and drug-resistant cancer cells. Subcutaneous and oral administration of 4Bb could effectively inhibit the growth of drug-resistant tumors in vivo, doubling the survival time of tumor bearing mice without side effects in normal tissues. Thus, our study offers an orally-available, structurally-novel PLK1 inhibitor for drug-resistant lung cancer therapy.

Biotin-tagged fluorescent probe for in situ visualization of γ-glutamyl transpeptidase in cancerous cells and tissues.

γ-Glutamyl transpeptidase (GGT), a cell-surface enzyme, is strongly implicated in mammalian malignancy growth and migration processes including human hepatocarcinogens. However, simply and conveniently detect of GGT on the cell membrane remains highly challenging. In this study, a biotin-tagged fluorescent probe Nap-biotin-glu was developed using glutamic acid, naphthalimide, and biotin as the reaction site, fluorescent reporter, and membrane-targeting group, which required only three steps. Colocalization fluorescence imaging and immunofluorescence analysis indicated that probe Nap-biotin-glu was successfully realized in situ visualizing of GGT on the cell membrane.Owing to the significant over-expressed GGT level in tumor, the probe was successfully applied to distinguish cancer tissues from adjacent normal tissues.

Engineered Artesunate-Naphthalimide Hybrid Dual Drug for Synergistic Multimodal Therapy against Experimental Murine Lymphoma.

Lymphoma can effectively be treated with a chemotherapy regimen that is associated with adverse side effects due to increasing drug resistance, so there is an emergent need for alternative small-molecule inhibitors to overcome the resistance that occurs in lymphoma management and overall increase the prognosis rate. A new series of substituted naphthalimide moieties conjugated via ester and amide linkages with artesunate were designed, synthesized, and characterized. In addition to the conjugates, to further achieve a theranostic molecule, FITC was incorporated via a multistep synthesis process. DNA binding studies of these selected derivatives by ultraviolet-visible (UV-vis), fluorescence spectroscopy, intercalating dye (EtBr, acridine orange)-DNA competitive assay, and minor groove binding dye Hoechst 33342-DNA competitive assay suggested that the synthesized novel molecules intercalated between the two strands of DNA due to its naphthalimide moiety and its counterpart artesunate binds with the minor groove of DNA. Napthalimide-artesunate conjugates inhibit the growth of lymphoma and induce apoptosis, including ready incorporation and reduction in cell viability. The remodeled drug has a significant tumoricidal effect against solid DL tumors developed in BALB/c mice in a dose-dependent manner. The novel drug appears to inhibit metastasis and increase the survival of the treated animals compared with untreated littermates.

Research Progress on Structure-Activity Relationship of 1,8-Naphthalimide DNA Chimeras Against Tumor.

The development of 1,8-naphthalimide derivatives as cell probes, DNA targeting agents, and anti-tumor drugs is one of the research hotspots in the field of medicine. Naphthalimide compounds are a kind of DNA embedder, which can change the topological structure of DNA by embedding in the middle of DNA base pairs, and then affect the recognition and action of topoisomerase on DNA. Aminofide and mitonafide are the first 2 drugs to undergo clinical trials. They have good DNA insertion ability, can embed DNA double-stranded structure, and induce topoisomerase II to cut part of pBR322DNA, but not yet entered the market due to their toxicity. In this paper, the design and structure-activity relationship of mononaphthalimide and bisaphthalimide compounds were studied, and the relationship between the structure of naphthalimide and anti-tumor activity was analyzed and discussed. It was found that a variety of structural modifications were significant in improving anti-tumor activity and reducing toxicity.

A bio-friendly biotin-coupled and azide-functionalized naphthalimide for real-time endogenous hydrogen sulfide analysis in living cells.

Hydrogen sulfide (H2S) is involved in various biological processes. Thereby, abnormal levels of H2S are reported to be related to various human diseases including cancer. Currently, many fluorescent probes are pioneered to detect H2S by taking advantages of naphthalimides' unique internal charge transfer (ICT) property. However, most probes often require a high content of organic solvents or surfactants, and are limited to the analysis of exogenous H2S treated externally in live cell studies, and have difficulties in analyzing endogenous H2S, thus limiting their practical use. In this study, we developed a bio-friendly biotin-coupled and azide-functionalized naphthalimide (1) as a fluorescent probe enabling real-time analysis of H2S in living system. Probe was able to provide a fluorescence at 545 nm via H2S-mediated azide reduction selectively without interference by biologically abundant constituents and pH effects. In a biological study using A549 cells, probe readily penetrated living cells without cytotoxicity, and unreacted probes showed almost no fluorescence, enabling real-time detection of H2S in living cells without requiring separate washing process. More importantly, under stimulation with various H2S inducers and inhibitors, probe was able to provide an effective fluorescence response against fluctuations in endogenous H2S, a key requirement for H2S studies. Probe 1 can be applied as a useful chemical tool and enables the analysis of H2S and the study of H2S-related cell functions in a variety of environments.

Bioimaging of labile lysosomal iron through naphthalimide-based fluorescent probe.

Lysosomal labile iron detection is immensely important as it is related to various diseases like Alzheimer's disease, Huntington's disease, Parkinson's disease, and cell apoptosis like ferroptosis. The fluorescent-based detection methods are preferred due to their sensitive, non-invasive, and spatial-temporal detection in biological samples. However, this remains a great challenge due to the lysosomal compartment being acidic alters the photophysical properties of the probe. Herein, we have rationally designed and synthesized multi-component naphthalimide-based fluorescent marker with preferred optical properties and bio-compatibility for selective detection of labile iron present in the lysosomal compartment. The synthesized probe was characterized structurally and optically by NMR, mass spectrometry, UV-visible, and fluorescence spectroscopy. The developed probe with an appropriate linking strategy turns out to be tolerant to fluorescence alternation in lysosomal pH. The probe exhibits great selectivity and high sensitivity for Fe(III) with limit of detection of 0.44 µM and is also able to detect Fenton-type reactions. Further, the probe has been successfully applied for lysosomal imaging and detecting labile Fe(III) present in the lysosomal lumen of the live cells.

Hook, Line, and Sinker! Spectroscopic Studies of Bi-Modular Mono- and Bis-1,8-naphthalimide-Ru(bpy)3-conjugates as DNA "Light Switches".

Bi-chromophoric ruthenium polypyridyl complexes comprising one or two nitro-1,8-naphthalimide groups are shown to be effective DNA binders with off-on light switching properties. The binding to DNA was investigated using a combination of studies such as UV-visible absorption and emission titrations, thermal denaturation, and circular dichroism spectroscopy. The DNA affinity was shown to be sensitive to both the linker length and the number of naphthalimides (one vs two) contained in these systems and binding constants ranging from 106 to 107 M-1 for salmon testes DNA. The strong DNA binding is attributed to the combination of naphthalimide intercalation and the electrostatic interaction of the ruthenium complex. Large emission enhancements from the metal to ligand charge transfer (MLCT) emission arising from the metal complex were observed upon DNA binding, which was attributed to the interruption of intramolecular electron transfer quenching processes. Moving the nitro substitution from the 4-position to the 3-position is found to result in modification of the DNA binding and the resulting optical properties. The off-on light switch phenomena reported demonstrate the potential of these complexes to act as DNA probes.

Rhodamine-thiourea Linked Naphthalimide Derivative to Image ATP in Mitochondria using Two Channels.

Adenosine 5'-triphosphate (ATP), synthesized in mitochondria, is an energy molecule in all living things. ATP not only serves as an energy source for protein synthesis and muscle contraction, but also as an important indicator for various diseases, such as Parkinson's disease, cardiovascular disease, and others. Accordingly, detection and sensing of ATP, especially in mitochondria, are important. In this study, a unique ring-opening process of rhodamine was coupled to recognition of ATP via introduction of a thiourea moiety, which was further linked to a naphthalimide group. A strong fluorescent emission at ∼580 nm was accompanied by a color change from colorless to pink upon addition of ATP at pH 7.4. Fluorescent probe 1 successfully imaged mitochondrial ATP with a Pearson's coefficient of 0.8. In addition, green emission from the naphthalimide moiety at ∼530 nm was observed without any change upon addition of ATP. This emission can be considered equivalent to an internal standard to utilize probe 1 as a dual-channel probe for ATP. Furthermore, probe 1 showed negligible cytotoxicity based on MTT assays.

Design of DNA Intercalators Based on 4-Carboranyl-1,8-Naphthalimides: Investigation of Their DNA-Binding Ability and Anticancer Activity.

In the present study, we continue our work related to the synthesis of 1,8-naphthalimide and carborane conjugates and the investigation of their anticancer activity and DNA-binding ability. For this purpose, a series of 4-carboranyl-1,8-naphthalimide derivatives, mitonafide, and pinafide analogs were synthesized using click chemistry, reductive amination, amidation, and Mitsunobu reactions. The calf thymus DNA (ct-DNA)-binding properties of the synthesized compounds were investigated by circular dichroism (CD), UV-vis spectroscopy, and thermal denaturation experiments. Conjugates 54-61 interacted very strongly with ct-DNA (∆Tm = 7.67-12.33 °C), suggesting their intercalation with DNA. They were also investigated for their in vitro effects on cytotoxicity, cell migration, cell death, cell cycle, and production of reactive oxygen species (ROS) in a HepG2 cancer cell line as well as inhibition of topoisomerase IIα activity (Topo II). The cytotoxicity of these eight conjugates was in the range of 3.12-30.87 µM, with the lowest IC50 value determined for compound 57. The analyses showed that most of the conjugates could induce cell cycle arrest in the G0/G1 phase, inhibit cell migration, and promote apoptosis. Two conjugates, namely 60 and 61, induced ROS production, which was proven by the increased level of 2'-deoxy-8-oxoguanosine in DNA. They were specifically located in lysosomes, and because of their excellent fluorescent properties, they could be easily detected within the cells. They were also found to be weak Topo II inhibitors.

Indomethacin-induced spectral responses of naphthalimide-based dyes to serum albumin: effects of substituent and spacer.

Four indomethacin-naphthalimide binaries with different proton receptors at 4-position of naphthalimide were designed and synthesized. N,N-Dimethylethylenediamine and N-methyl piperazine were served as proton receptors as well as solubility regulators. Indomethacin, an inhibitor for cyclooxygenase-2 overexpressed on cancer cells, was connected at the imine N through different spacers. The attachment of indomethacin significantly quenched the fluorescence of all compounds with obvious red-shift in the absorption maxima due to the strong photo-induced electron transfer process of the folded-state. Human serum albumin (HSA) triggered about 15-fold fluorescence enhancements of DMN-IMC-5 with 30 nm blue-shift. However, it caused much smaller fluorescence increments of other compounds, suggesting that indomethacin, the linker and proton receptor play critical roles in HSA identification. Fluorescence bioimaging results show that indomethacin enables the naphthalimide-based compounds to fluorescent imaging living cells. Molecular docking reveals that the introduction of indomethacin improved the binding affinity of the dyes to HSA.

Dual-Targeting into the Mitochondria of Cancer Cells for Ratiometric Investigation of the Dynamic Fluctuation of Sulfur Dioxide and Formaldehyde with Two-Photon Integrated Semiconducting Polymer Dots.

Mitochondrial sulfur dioxide (SO2) and formaldehyde (FA) in cancer cells serve as important signal molecules in mediating multiple physiological and pathological activities. Accurate monitoring of the dynamic fluctuation of SO2 and FA in the mitochondria of cancer cells is important for insight into their relationships and functions in cancer, understanding cancer mechanism, and the role of mitochondrial homeostasis in cancer invasion and metastasis. Herein, a novel integrated two-photon semiconducting polymer dot (BF@Pdots) with dual-targeting (cancer cells and mitochondrial) and dual-emission in green and red regions, which is rationally designed through a four-step engineering strategy by using two newly synthesized functionalized polymers PFNA and FD-PSMA as precursors, has been developed for accurate tracking of the dynamic variation of SO2 and FA in the mitochondria of cancer cells. The sensing mechanism is on the basis of the fluorescence resonance energy transfer (FRET) process in BF@Pdots tuned by the reversible Michael addition reaction between the sensing-groups and SO2 (or FA). The integrated BF@Pdots nanoprobes display excellent performances in the accurate detection of the dynamic fluctuation of SO2 and FA such as precise positioning in the mitochondria of cancer cells, self-calibrating ratiometric, two-photon emission with long wavelength excitation, and fast reversible response. The BF@Pdots nanoprobes are also applied to the ratiometric detection of the dynamic fluctuation of exogenous and endogenous SO2 and FA in the mitochondria of cancer cells for the first time with satisfactory results. Taken together, this work will provide an attractive way to develop versatile integrated Pdots-based fluorescent probes through flexible molecular engineering for applications in accurate imaging of biomolecules in living systems.

A novel reversible fluorescent probe based on naphthalimide for sequential detection of aluminum (Al3+) and fluoride (F-) ions and its applications.

A new Schiff base fluorescent probe NBP derived from the one-step condensation strategy of 2-butyl-6-hydroxy-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinoline-5-carbaldehyde and N-(2-(hydrazinecarbonyl)phenyl)benzamide was synthesized and characterized. NBP exhibited high selectivity toward Al3+ along with naked-eye color changes and prominent fluorescence enhancement. The limit of detection (LOD) of NBP toward Al3+ was detected to be 80 nM. The binding ratio of NBP with Al3+ ions was obtained as 1 : 2 on the basis of Job's plot with the association constant Ka value of 4.22 × 1010 M-1/2. The plausible complexation mechanism of NBP toward Al3+ ions was validated by the density functional theory (DFT) and IR spectrum. In addition, in situ formed "NBP + Al3+" could be utilized as the second sensor for selective recognition of F-via fluorescence quenching with a low detection limit (44 nM). Furthermore, the cell imaging experiments of probe NBP in HeLa cells have successfully demonstrated that NBP could serve as an indicator for monitoring Al3+ ions in living cells. On top of that, NBP could be used to prepare simple test paper strips for quickly and qualitatively detecting a trace amount of Al3+ ions in a visible manner.

Reactive Oxygen Species Mediate 6c-Induced Mitochondrial and Lysosomal Dysfunction, Autophagic Cell Death, and DNA Damage in Hepatocellular Carcinoma.

Increasing the level of reactive oxygen species (ROS) in cancer cells has been suggested as a viable approach to cancer therapy. Our previous study has demonstrated that mitochondria-targeted flavone-naphthalimide-polyamine conjugate 6c elevates the level of ROS in cancer cells. However, the detailed role of ROS in 6c-treated cancer cells is not clearly stated. The biological effects and in-depth mechanisms of 6c in cancer cells need to be further investigated. In this study, we confirmed that mitochondria are the main source of 6c-induced ROS, as demonstrated by an increase in 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and MitoSox fluorescence. Compound 6c-induced mitochondrial ROS caused mitochondrial dysfunction and lysosomal destabilization confirmed by absolute quantitation (iTRAQ)-based comparative proteomics. Compound 6c-induced metabolic pathway dysfunction and lysosomal destabilization was attenuated by N-acetyl-L-cysteine (NAC). iTRAQ-based comparative proteomics showed that ROS regulated the expression of 6c-mediated proteins, and treatment with 6c promoted the formation of autophagosomes depending on ROS. Compound 6c-induced DNA damage was characterized by comet assay, p53 phosphorylation, and γH2A.X, which was diminished by pretreatment with NAC. Compound 6c-induced cell death was partially reversed by 3-methyladenine (3-MA), bafilomycin (BAF) A1, and NAC, respectively. Taken together, the data obtained in our study highlighted the involvement of mitochondrial ROS in 6c-induced autophagic cell death, mitochondrial and lysosomal dysfunction, and DNA damage.

Photodiagnosis and photodynamic effects of bacteriochlorin-naphthalimide conjugates on tumor cells and mouse model.

Photo-induced cytotoxicity and antitumor activity of a series of dual function agents for photodynamic therapy (PDT) and fluorescent imaging based on bacteriochlorin photosensitizer conjugated with various naphthalimide fluorophores was studied in vitro using murine tumor cells of S37 sarcoma and in vivo on mice bearing murine S37 sarcoma. Upon irradiation at the absorption maximum of the photosensitizer, the activity of conjugates was as high as in the case of individual bacteriochlorin, while an additional excitation of the naphthalimide fragment led to an increase in the PDT efficacy due to resonance energy transfer from the fluorophore to photosensitizer. The fluorescence contrast and specific cytotoxic activity measurements indicate that the conjugate of bacteriochlorin with 3,4-dimethoxestyrene-substituted naphthalimide is the most promising agent for the application as theranostic in PDT.

Simultaneous inhibition of planktonic and biofilm bacteria by self-adapting semiconducting polymer dots.

Biofilm infections present an enormous challenge in today's healthcare settings. Currently, pH-switchable antibacterial agents are being developed to eradicate biofilms. However, most pH-switchable antibacterial agents are less lethal to planktonic bacteria under neutral conditions, and cannot prevent the dispersed bacteria from seeding acute infection again. Herein, this work reports the applications of semiconducting polymer dots (Pdots) with a double adhesion mechanism in imaging and inhibiting bacteria inside (weak acidic conditions) and outside (neutral conditions) biofilms. Clew-like Pdots were prepared by covalently linking phenylboronic acid (PBA) and pH-responsive naphthalimide (NA) ramification in semiconducting polymers. Under neutral conditions, the Pdots combined with bacteria through the formation of boronate esters between PBA and diols. Under weakly acidic conditions, the partial borate bond fractured, and the Pdots adhered onto the bacterial surface through the positively charged NA in Pdots. Furthermore, the Pdots display negligible toxicity to mammalian cells and tissues. More importantly, the Pdots can selectively damage the bacterial membrane and inhibit bacteria in vivo. This work highlights the feasibility of using semiconducting Pdots to image and inhibit bacteria inside and outside biofilms, which represents a highly effective strategy to cope with biofilm infections.

A novel naphthalimide derivative reduces platelet activation and thrombus formation via suppressing GPVI.

Naphthalimide derivatives have multiple biological activities, including antitumour and anti-inflammatory activities. We previously synthesized several naphthalimide derivatives; of them, compound 5 was found to exert the strongest inhibitory effect on human DNA topoisomerase II activity. However, the effects of naphthalimide derivatives on platelet activation have not yet been investigated. Therefore, the mechanism underlying the antiplatelet activity of compound 5 was determined in this study. The data revealed that compound 5 (5-10 µM) inhibited collagen- and convulxin- but not thrombin- or U46619-mediated platelet aggregation, suggesting that compound 5 is more sensitive to the inhibition of glycoprotein VI (GPVI) signalling. Indeed, compound 5 could inhibit the phosphorylation of signalling molecules downstream of GPVI, followed by the inhibition of calcium mobilization, granule release and GPIIb/IIIa activation. Moreover, compound 5 prevented pulmonary embolism and prolonged the occlusion time, but tended to prolong the bleeding time, indicating that it can prevent thrombus formation but may increase bleeding risk. This study is the first to demonstrate that the naphthalimide derivative compound 5 exerts antiplatelet and antithrombotic effects. Future studies should modify compound 5 to synthesize more potent and efficient antiplatelet agents while minimizing bleeding risk, which may offer a therapeutic potential for cardiovascular diseases.

Mechanism of BIP-4 mediated inhibition of InsP3Kinase-A.

Overexpression of the neuronal InsP3kinase-A increases malignancy of different tumor types. Since InsP3kinase-A highly selectively binds Ins(1,4,5)P3, small molecules competing with Ins(1,4,5)P3 provide a promising approach for the therapeutic targeting of InsP3kinase-A. Based on this consideration, we analyzed the binding mechanism of BIP-4 (2-[3,5-dimethyl-1-(4-nitrophenyl)-1H-pyrazol-4-yl]-5, 8-dinitro-1H-benzo[de]isoquinoline-1,3(2H)-dione), a known competitive small-molecule inhibitor of Ins(1,4,5)P3. We tested a total of 80 BIP-4 related compounds in biochemical assays. The results of these experiments revealed that neither the nitrophenyl nor the benzisochinoline group inhibited InsP3kinase-A activity. Moreover, none of the BIP-4 related compounds competed for Ins(1,4,5)P3, demonstrating the high selectivity of BIP-4. To analyze the inhibition mechanism of BIP-4, mutagenesis experiments were performed. The results of these experiments suggest that the nitro groups attached to the benzisochinoline ring compete for binding of Ins(1,4,5)P3 while the nitrophenyl group is associated with amino acids of the ATP-binding pocket. Our results now offer the possibility to optimize BIP-4 to design specific InsP3Kinase-A inhibitors suitable for therapeutic targeting of the enzyme.