Synthesis of acridone derivatives via heterologous expression of a plant type III polyketide synthase in Escherichia coli.

BACKGROUND: Acridone alkaloids are heterocyclic compounds that exhibit a broad-range of pharmaceutical and chemotherapeutic activities, including anticancer, antiviral, anti-inflammatory, antimalarial, and antimicrobial effects. Certain plant species such as Citrus microcarpa, Ruta graveolens, and Toddaliopsis bremekampii synthesize acridone alkaloids from anthranilate and malonyl-CoA. RESULTS: We synthesized two acridones in Escherichia coli. Acridone synthase (ACS) and anthraniloyl-CoA ligase genes were transformed into E. coli, and the synthesis of acridone was examined. To increase the levels of endogenous anthranilate, we tested several constructs expressing proteins involved in the shikimate pathway and selected the best construct. To boost the supply of malonyl-CoA, genes coding for acetyl-coenzyme A carboxylase (ACC) from Photorhabdus luminescens were overexpressed in E. coli. For the synthesis of 1,3-dihydroxy-10-methylacridone, we utilized an N-methyltransferase gene (NMT) to supply N-methylanthranilate and a new N-methylanthraniloyl-CoA ligase. After selecting the best combination of genes, approximately 17.3 mg/L of 1,3-dihydroxy-9(10H)-acridone (DHA) and 26.0 mg/L of 1,3-dihydroxy-10-methylacridone (NMA) were synthesized. CONCLUSIONS: Two bioactive acridone derivatives were synthesized by expressing type III plant polyketide synthases and other genes in E. coli, which increased the supplement of substrates. This study showed that is possible to synthesize diverse polyketides in E. coli using plant polyketide synthases.

Probing the Hydrogen Bond Involving Acridone Trapped in a Hydrophobic Biological Nanocavity: Integrated Spectroscopic and Docking Analyses.

Spectroscopic analyses reveal that acridone (AD) penetrates through the structure and enters the hydrophobic cavity of the protein ß-lactoglobulin (ßLG). Although the protein contains two tryptophan (Trp) residues, AD interacts with only one (Trp-19), which is authenticated by the appearance of a single isoemissive point in TRANES. Alteration in the secondary structure of the protein while AD pierces through ßLG is evident from the circular dichroism spectroscopic study. The ground-state interaction between AD and ßLG is proven from the UV-vis spectroscopic study and the static nature of quenching of intrinsic fluorescence of the protein by the ligand. The steady-state fluorescence study in varied temperatures indicates the involvement of hydrogen bonding in the ligand-protein interaction. Further, the time-resolved fluorescence anisotropy study gives a hint of the presence of a hydrogen bond in AD-ßLG interaction, which possibly involves the rotamers of Trp-19. In fact, the idea of involvement of rotamers of Trp-19 is obtained from the increase in fluorescence lifetime of ßLG in the presence of AD. The docking study agrees to the involvement of hydrogen bonding in AD-ßLG interaction. The direct evidence of hydrogen bonding between Trp and AD is obtained from the laser flash photolysis studies where the signature of formation of ADH• and Trp• through hydrogen abstraction between Trp and AD, loosely bound through hydrogen bonding, gets prominence. Thus, binding of AD to ßLG involves hydrogen bonding in a hydrophobic pocket of the protein.

A competent synthesis and efficient anti-inflammatory responses of isatinimino acridinedione moiety via suppression of in vivo NF-κB, COX-2 and iNOS signaling.

A potent Nonsterodial Anti-inflammatory Drug (NSAID) candidates has been conceived and built by an assembly of a hydrophilic, fluorescent and COX-2 inhibiting units in the same molecule. The isatinimino-acridinedione core (TM-7) was achieved in a simple three step synthetic procedure viz (i) a multicomponent reaction between dimedone, aldehyde and amine to furnish the nitroacridinedione (4), (ii) reduction step and (iii) schiff's-base condensation with isatin. The excellent anti-inflammatory pharmacological efficiency of the drug was established by in vivo biological experiments. Accordingly, it was found that the treatment with the synthesized isatinimino analogues (dosage: 30 mg/kg) inhibited protein expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NF-κB) as well as production of prostaglandin E2 (PGE2), nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and interleukin-6 (IL-6) levels induced by carrageenan. Further, a comparative molecular modeling analysis of TM-7 carried out with the crystal structure of aspirin acetylated human COX-2 suggested effectively binding and efficient accommodation inside the active site's gorge.

Multiscale time-resolved fluorescence study of a glycogen phosphorylase inhibitor combined with quantum chemistry calculations.

A fluorescence study of N1-(ß-d-glucopyranosyl)-N4-[2-acridin-9(10H)-onyl]-cytosine (GLAC), the first fluorescent potent inhibitor of glycogen phosphorylase (GP), in neutral aqueous solution, is presented herein. Quantum chemistry (TD-DFT) calculations show the existence of several conformers both in the ground and first excited states. They result from rotations of the acridone and cytosine moieties around an NH bridge which may lead to the formation of non-emitting charge-transfer states. The fingerprints of various conformers have been detected by time-resolved fluorescence spectroscopy (fluorescence upconversion and time-correlated single photon counting) and identified using as criteria their energy, polarization and relative population resulting from computations. Such an analysis should contribute to the design of new GP inhibitors with better fluorescence properties, suitable for imaging applications.

Synthesis and evaluation of 1,2,3,4-tetrahydro-1-acridone analogues as potential dual inhibitors for amyloid-beta and tau aggregation.

Amyloid-ß (Aß) and tau protein are two crucial hallmarks in Alzheimer's disease (AD). Their aggregation forms are thought to be toxic to the neurons in the brain. A series of new 1,2,3,4-tetrahydro-1-acridone analogues were designed, synthesized, and evaluated as potential dual inhibitors for Aß and tau aggregation. In vitro studies showed that compounds 25-30 (20 µM) with N-methylation of the quinolone ring effectively inhibited Aß1-42 aggregation by 84.7%-99.5% and tau aggregation by 71.2%-101.8%. Their structure-activity relationships are discussed. In particular, 30 could permeate the blood-brain barrier, bind to Aß1-42 and tau, inhibit Aß1-42 ß-sheets formation, and prevent tau aggregation in living cells.

Acridone Alkaloids.

There have been substantial developments in the chemistry and biology of the acridone alkaloids in the 16years since the topic was last reviewed in this series of monographs (2000). The present survey covers the literature from mid-1999 to 2016. A brief overview of the biosynthesis of acridone alkaloids is followed by details of the occurrence and characterization of known alkaloids from new sources, and of novel alkaloids. The classes covered include simple acridone alkaloids, C-prenylacridones, furo[3,2-b]- and furo[2,3-c]acridones, pyrano[3,2-b]- and pyrano[2,3-c]acridones, and dimeric alkaloids containing acridone moieties. Syntheses of acridone alkaloids and certain analogs reported during the review period are comprehensively covered. The final section summarizes aspects of their bioactivity, including cytotoxicity and anticancer activity, antimicrobial and antiparasitic properties, and enzyme inhibition. The chapter concludes with a brief description of important bioactive synthetic analogs.

Potent acetylcholinesterase inhibitors: Synthesis, biological assay and docking study of nitro acridone derivatives.

The reaction of o-halobenzoic acid with aniline derivatives and their subsequent cyclization reaction yielded the acridone derivatives. The series of nitro acridone derivatives were prepared by Ullmann condensation in presence of copper as catalyst and were characterized by FTIR, (1)H, (13)C NMR and mass spectra. The structure of 5-nitro-(2-phenyl amino) benzoic acid (4) was confirmed by X-ray crystallography and was found to crystallize in P21/c space group. The in vitro efficacy of the compounds for their acetylcholinesterase (AChE) and antimicrobial inhibitory activities have been evaluated against the standard drugs Ampicillin and Gentamicin against Gram positive and Gram negative bacteria. 1,7-Dinitroacridone was found to be the most potent AChE inhibitor (IC50=0.22µM). Moreover, the compounds have been screened for their antioxidant activity using the DPPH assay. Also, docking study results were found to be in good agreement with the results obtained through in vitro experiments. The docking study further predicted possible binding conformation.

Molecular design, synthesis and biological research of novel pyridyl acridones as potent DNA-binding and apoptosis-inducing agents.

A series of novel pyridyl acridone derivatives comprised of a pseudo-five-cyclic system to extend the π-conjugated acridone chromophore, were designed and synthesized as potent DNA binding antitumor compounds. Most synthesized compounds displayed good activity against human leukemia K562 cells in MTT tests, with compound 6d exhibiting the highest activity with IC50 value at 0.46 µM. Moreover, 6d showed potent activities against solid tumor cell lines (0.16-3.79 µM). Several experimental studies demonstrated that the antitumor mode of action of compound 6d involves DNA intercalation, topoisomerase I inhibition, and apoptosis induction through the mitochondrial pathway. In summary, compound 6d represents a novel and promising lead structure for the development of new potent anticancer DNA-binding agents.

Cloning and structure-function analyses of quinolone- and acridone-producing novel type III polyketide synthases from Citrus microcarpa.

Two novel type III polyketide synthases, quinolone synthase (QNS) and acridone synthase (ACS), were cloned from Citrus microcarpa (Rutaceae). The deduced amino acid sequence of C. microcarpa QNS is unique, and it shared only 56-60% identities with C. microcarpa ACS, Medicago sativa chalcone synthase (CHS), and the previously reported Aegle marmelos QNS. In contrast to the quinolone- and acridone-producing A. marmelos QNS, C. microcarpa QNS produces 4-hydroxy-N-methylquinolone as the "single product" by the one-step condensation of N-methylanthraniloyl-CoA and malonyl-CoA. However, C. microcarpa ACS shows broad substrate specificities and produces not only acridone and quinolone but also chalcone, benzophenone, and phloroglucinol from 4-coumaroyl-CoA, benzoyl-CoA, and hexanoyl-CoA, respectively. Furthermore, the x-ray crystal structures of C. microcarpa QNS and ACS, solved at 2.47- and 2.35-Å resolutions, respectively, revealed wide active site entrances in both enzymes. The wide active site entrances thus provide sufficient space to facilitate the binding of the bulky N-methylanthraniloyl-CoA within the catalytic centers. However, the active site cavity volume of C. microcarpa ACS (760 Å(3)) is almost as large as that of M. sativa CHS (750 Å(3)), and ACS produces acridone by employing an active site cavity and catalytic machinery similar to those of CHS. In contrast, the cavity of C. microcarpa QNS (290 Å(3)) is significantly smaller, which makes this enzyme produce the diketide quinolone. These results as well as mutagenesis analyses provided the first structural bases for the anthranilate-derived production of the quinolone and acridone alkaloid by type III polyketide synthases.

A template model for studying anticancer drug efflux transporter inhibitors in vitro.

Efflux transporters play an important role in drug absorption and also in multidrug resistance. ABCG2 (BCRP) is an efflux transporter conferring cross-resistance to mitoxantrone (Mit), irinotecan (CPT11), and its active metabolite SN38. MBLI87, a new ABCG2 inhibitor has proven its efficacy against ABCG2-mediated efflux in vitro and in vivo. This work aimed at modeling and quantifying the cellular interaction between MBLI87 and different substrates using a mechanistic template model. An in vitro competition experiment study was carried out with HEK293 cells overexpressing ABCG2 exposed to fixed concentrations of substrates (Mit, CPT11, SN38) and to MBLI87 at several concentration levels. A nonlinear mixed-effects transport inhibition model was developed to fit intracellular drug concentrations. In this model, drugs cross the cell membrane through passive diffusion, active drug efflux is ABCG2 mediated, interaction between substrates and inhibitor occurs within the transporter. The interaction was found to be noncompetitive. The MBLI87 Ki was estimated to 141 nm for Mit, 289 nm for CPT11, and 1160 nm for SN38. The ratio of intrinsic transport clearance divided by diffusion clearance was estimated to 2.5 for Mit, 1.01 for CPT11, and 5.4 for SN38. The maximal increase in the intracellular substrate concentration that is possible to achieve by inhibition of the transporter was estimated to 1.5 for Mit, 0.1 for CPT11, and 4.4 for SN38. This mechanistic template model describes both drug accumulation and cellular transport, and the mixed-effects approach allows an estimation of intra- and interassay variability. This model is of great interest to study cytotoxic cellular pharmacokinetics.

Discovery of dual function acridones as a new antimalarial chemotype.

Preventing and delaying the emergence of drug resistance is an essential goal of antimalarial drug development. Monotherapy and highly mutable drug targets have each facilitated resistance, and both are undesirable in effective long-term strategies against multi-drug-resistant malaria. Haem remains an immutable and vulnerable target, because it is not parasite-encoded and its detoxification during haemoglobin degradation, critical to parasite survival, can be subverted by drug-haem interaction as in the case of quinolines and many other drugs. Here we describe a new antimalarial chemotype that combines the haem-targeting character of acridones, together with a chemosensitizing component that counteracts resistance to quinoline antimalarial drugs. Beyond the essential intrinsic characteristics common to deserving candidate antimalarials (high potency in vitro against pan-sensitive and multi-drug-resistant Plasmodium falciparum, efficacy and safety in vivo after oral administration, inexpensive synthesis and favourable physicochemical properties), our initial lead, T3.5 (3-chloro-6-(2-diethylamino-ethoxy)-10-(2-diethylamino-ethyl)-acridone), demonstrates unique synergistic properties. In addition to 'verapamil-like' chemosensitization to chloroquine and amodiaquine against quinoline-resistant parasites, T3.5 also results in an apparently mechanistically distinct synergism with quinine and with piperaquine. This synergy, evident in both quinoline-sensitive and quinoline-resistant parasites, has been demonstrated both in vitro and in vivo. In summary, this innovative acridone design merges intrinsic potency and resistance-counteracting functions in one molecule, and represents a new strategy to expand, enhance and sustain effective antimalarial drug combinations.

Investigation of DNA-binding properties of organic molecules using quantitative structure-activity relationship (QSAR) models.

Due to the great potential of DNA as a receptor, many classes of synthetic and naturally occurring molecules exert their anticancer activities through DNA-binding. In the field of antitumor DNA-binding agents, a number of acridine and anthracycline derivatives are in the market as chemotherapeutic agents. However, the clinical application of such classes of compounds has encountered problems such as multi-drug resistance and secondary and/or collateral effects. Thus, there has been increasing interest in discovering and developing small molecules that are capable of DNA-binding, which will be expected to be used either in place of or in conjunction with, the existing compounds. The interest in the application of the QSAR paradigm has steadily increased in recent decades and we hope it may be useful in the design and development of DNA-binding molecules as new anticancer agents. In the present review, an attempt has been made to understand the DNA-binding properties of different compound series and discussed using 27 QSAR models, which reveal a number of interesting points. The most important determinants for the activity in these models are Hammett electronic (sigma and sigma+), hydrophobic, molar refractivity, and Sterimol width parameters.

Interactions of antitumor triazoloacridinones with DNA.

Triazoloacridinones (TA) are a new group of potent antitumor compounds, from which the most active derivative, C-1305, has been selected for extended preclinical trials. This study investigated the mechanism of TA binding to DNA. Initially, for selected six TA derivatives differing in chemical structures as well as cytotoxicity and antitumor activity, the capability of noncovalent DNA binding was analyzed. We showed that all triazoloacridinones studied stabilized the DNA duplex at a low-concentration buffer but not at a salt concentration corresponding to that in cells. DNA viscometric studies suggested that intercalation to DNA did not play a major role in the mechanism of the cytotoxic action of TA. Studies involving cultured cells revealed that triazoloacridinone C-1305 after previous metabolic activation induced the formation of interstrand crosslinks in DNA of some tumor and fibroblast cells in a dose dependent manner. However, the detection of crosslink formation was possible only when the activity of topoisomerase II in cells was lowered. Furthermore, it was impossible to validate the relevance of the ability to crosslink DNA to biological activity of TA derivatives.