Modification of bacterial microcompartments with target biomolecules via post-translational SpyTagging.

Bacterial microcompartments (BMCs) are proteinaceous organelle-like structures formed within bacteria, often encapsulating enzymes and cellular processes, in particular, allowing toxic intermediates to be shielded from the general cellular environment. Outside of their biological role they are of interest, through surface modification, as potential drug carriers and polyvalent antigen display scaffolds. Here we use a post-translational modification approach, using copper free click chemistry, to attach a SpyTag to a target protein molecule for attachment to a specific SpyCatcher modified BMC shell protein. We demonstrate that a post-translationally SpyTagged material can react with a SpyCatcher modified BMC and show its presence on the surface of BMCs, enabling future investigation of these structures as polyvalent antigen display scaffolds for vaccine development. This post-translational 'click' methodology overcomes the necessity to genetically encode the SpyTag, avoids any potential reduction in expression yield and expands the scope of SpyTag/SpyCatcher vaccine scaffolds to form peptide epitope vaccines and small molecule delivery agents.

Activity of Aromathecins against African Trypanosomes.

African sleeping sickness is responsible for thousands of deaths annually, and new therapeutics are needed. This study evaluated aromathecins, experimental inhibitors of mammalian topoisomerase IB, against Trypanosoma brucei African trypanosomes. The compounds had selectively toxic antiparasitic potency, in situ poisoning activity against the phylogenetically unique topoisomerase in these parasites, and a representative compound intercalated into DNA with micromolar affinity. DNA intercalation and topoisomerase poisoning may contribute to the antitrypanosomal activity of aromathecins.

Synthesis and biological evaluation of indenoisoquinolines that inhibit both tyrosyl-DNA phosphodiesterase I (Tdp1) and topoisomerase I (Top1).

Tyrosyl-DNA phosphodiesterase I (Tdp1) plays a key role in the repair of damaged DNA resulting from the topoisomerase I (Top1) inhibitor camptothecin and a variety of other DNA-damaging anticancer agents. This report documents the design, synthesis, and evaluation of new indenoisoquinolines that are dual inhibitors of both Tdp1 and Top1. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures were used to establish structure-activity relationships. The potencies of the indenoisoquinolines against Tdp1 ranged from 5 µM to 111 µM, which places the more active compounds among the most potent known inhibitors of this target. The cytotoxicity mean graph midpoints ranged from 0.02 to 2.34 µM. Dual Tdp1-Top1 inhibitors are of interest because the Top1 and Tdp1 inhibitory activities could theoretically work synergistically to create more effective anticancer agents.

Identification, synthesis, and biological evaluation of the metabolites of 3-amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36), a promising rexinoid lead compound for the development of cancer chemotherapeutic and chemopreventive agents.

Activation of the retinoid X receptor (RXR), which is involved in cell proliferation, differentiation, and apoptosis, is a strategy for cancer chemotherapy and chemoprevention, and 3-amino-6-(3'-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6-36) (3) is among the few RXR ligands known. The presently reported studies of 3 include its binding to human plasma proteins, metabolic stability using human liver microsomes, metabolism by human liver microsomes and hepatocytes, and in vivo disposition in rat serum, liver, and mammary tissue. Compound 3 was 75% bound to human plasma proteins, and its metabolic stability was much greater than propranolol. One phase I metabolite was formed by human liver microsomes, seven phase I and II metabolites were formed by human hepatocytes, and five metabolites were detected in rat serum and liver after oral administration. The putative metabolites predicted using LC-MS-MS were synthesized to confirm their structures and to provide sufficient material for investigation of induction of RXRE transcriptional activity and inhibition of NFκB.

Synthesis and biological evaluation of the first dual tyrosyl-DNA phosphodiesterase I (Tdp1)-topoisomerase I (Top1) inhibitors.

Substances with dual tyrosyl-DNA phosphodiesterase I-topoisomerase I inhibitory activity in one low molecular weight compound would constitute a unique class of anticancer agents that could potentially have significant advantages over drugs that work against the individual enzymes. The present study demonstrates the successful synthesis and evaluation of the first dual Top1-Tdp1 inhibitors, which are based on the indenoisoquinoline chemotype. One bis(indenoisoquinoline) had significant activity against human Tdp1 (IC(50) = 1.52 ± 0.05 µM), and it was also equipotent to camptothecin as a Top1 inhibitor. Significant insights into enzyme-drug interactions were gained via structure-activity relationship studies of the series. The present results also document the failure of the previously reported sulfonyl ester pharmacophore to confer Tdp1 inhibition in this indenoisoquinoline class of inhibitors even though it was demonstrated to work well for the steroid NSC 88915 (7). The current study will facilitate future efforts to optimize dual Top1-Tdp1 inhibitors.

Long-term risk of intraocular pressure elevation and glaucoma escalation after deep anterior lamellar keratoplasty.

BACKGROUND: To determine the prevalence and severity of intraocular pressure changes after deep anterior lamellar keratoplasty and its effect on visual function. DESIGN: Retrospective cohort study. PARTICIPANTS: All patients undergoing deep anterior lamellar keratoplasty procedures at a tertiary referral centre (Leeds University Hospitals, UK) using a manual dissection technique. METHODS: Case note review of all cases between the 1st of January 2000 and the 31st of December 2005. MAIN OUTCOME MEASURES: Raised intraocular pressure, glaucoma incidence or escalation. RESULTS: Data were collected for 69 of eyes of 58 patients. The mean period of follow-up was 54.9 months (median 60, range 6-95 months). The initial diagnosis was keratoconus in 56 cases (81%), corneal scarring in four cases (5.8%), herpes simplex keratitis in four cases (5.8%), lattice dystrophy in four cases (5.8%) and one case of corneal dermoid. Temporarily intraocular pressure was thought to be related to topical steroid use occurred in 12 (17%) cases. Ocular hypertension requiring treatment occurred in three eyes. None of these eyes had progressive disc changes or visual field defects suggestive of glaucoma, and all had well-controlled intraocular pressure on topical, single-drug therapy. Overall, there was a small insignificant rise in intraocular pressure after deep anterior lamellar keratoplasty (P = 0.11). CONCLUSIONS: The long-term risk of glaucoma following deep anterior lamellar keratoplasty using the manual dissection technique appears to be low. Ocular hypertension after deep anterior lamellar keratoplasty is infrequent and can be controlled on topical medication alone.

7-azaindenoisoquinolines as topoisomerase I inhibitors and potential anticancer agents.

A series of 7-azaindenoisoquinoline topoisomerase I (Top1) inhibitors have been prepared to investigate the effect of increased electron affinity of the aromatic system on the ability to stabilize the Top1-DNA cleavage complex. Ab initio calculations suggest that introduction of nitrogen into the aromatic system of the indenoisoquinolines would facilitate charge transfer complex formation with DNA, thus improving the π-π stacking interactions. The present study shows that 7-azaindenoisoquinolines demonstrate improved water solubility without any decrease in Top1 inhibitory activity or cytotoxicity. Analysis of the biological results reveals that smaller lactam ring substituents enable intercalation into both free DNA and Top1-DNA cleavage complex, whereas larger substituents only allow binding to the cleavage complex but not free DNA. Free DNA binding suppresses Top1-catalyzed DNA cleavage at high drug concentrations, whereas DNA cleavage and inhibition of religation occurs at low drug concentration.

Alcohol-, diol-, and carbohydrate-substituted indenoisoquinolines as topoisomerase I inhibitors: investigating the relationships involving stereochemistry, hydrogen bonding, and biological activity.

The DNA-relaxing enzyme topoisomerase I (Top1) can be inhibited by heterocyclic compounds such as indolocarbazoles and indenoisoquinolines. Carbohydrate and hydroxyl-containing side chains are essential for the biological activity of indolocarbazoles. The current study investigated how similar functionalities could be "translated" to the indenoisoquinoline system and how stereochemistry and hydrogen bonding affect biological activity. Herein is described the preparation and assay of indenoisoquinolines substituted with short-chain alcohols, diols, and carbohydrates. Several compounds (including those derived from sugars) display potent Top1 poisoning and antiproliferative activities. The Top1 poisoning activity of diol-substituted indenoisoquinolines is dependent upon stereochemistry. Although the effect is striking, molecular modeling and docking studies do not indicate any reason for the difference in activity due to similar calculated interactions between the ligand and Top1-DNA complex and ambiguity about the binding mode. A stereochemical dependence was also observed for carbohydrate-derived indenoisoquinolines. Although similar trends were observed in other classes of Top1 inhibitors, the exact nature of this effect has yet to be elucidated.

Induction of retinoid X receptor activity and consequent upregulation of p21WAF1/CIP1 by indenoisoquinolines in MCF7 cells.

Retinoid X receptor (RXR) has been targeted for the chemoprevention and treatment of cancer. To discover potential agents acting through RXRs, we utilized an RXR response element (RXRE)-luciferase reporter gene assay. Following extensive screening, 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride (AM6-36) was found to induce RXRE-luciferase activities. AM6-36 inhibited COX-2 expression and anchorage-independent growth with 12-O-tetradecanoylphorbol 13-acetate-stimulated JB6 Cl41 cells, induced the expression of CD38 in HL-60 cells, and attenuated the growth of N-methyl-N-nitrosourea-induced mammary tumors in rats. Consistent with other reports describing the antiproliferative effects of RXR agonists in breast cancers, AM6-36 showed growth inhibition with cultured MCF7 breast cancer cells, accompanied by G(2)/M-phase arrest at lower concentrations and enhanced S-phase arrest at higher concentrations. On the basis of DNA microarray analysis, AM6-36 upregulated the expression of CDKN1A, a target gene of RXR, by 35-fold. In accord with this response, the expression of the corresponding protein, p21(WAF1/CIP1), was increased in the presence of AM6-36. Induction of p21 by AM6-36 was abrogated following transient knockdown of RXRα, demonstrating that the effect of AM6-36 on the expression of p21 is closely related to modulation of RXRα transcriptional activity. Intestinal permeability was suggested with Caco-2 cells and limited metabolism resulted when AM6-36 was incubated with human liver microsomes. Oral administration with rats resulted in 0.8 µg/mL, 4.3 µg/g, and 0.3 µg/g in serum, liver, and mammary gland, respectively. In sum, these data suggest that AM6-36 is a promising lead for the treatment or prevention of breast cancer and provide a strong rationale for testing in more advanced antitumor systems.

Cancer chemopreventive potential of aromathecins and phenazines, novel natural product derivatives.

In the search for agents with cancer chemopreventive potential, 14-chloromethyl-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (compound 1), originally synthesized as a potential topoisomerase I inhibitor, and 2,4-dibromo-1-hydroxyphenazine (compound 2), an analog of a substance found in the marine bacteria Streptomyces CNS284, were found to significantly enhance NADP(H):quinone oxidoreductase 1 (QR1), glutathione S-transferase (GST), and glutathione (GSH) levels in cell culture. However, following a short-term absorption study, analyses of livers from the treatment groups did not reveal a significant increase in QR1 or GST activity, or GSH levels. This was consistent with RT-PCR analyses of tissue samples. The compounds were absorbed, as judged by LC/MS analyses of serum and tissue samples, although levels were well below the concentrations required to mediate in vitro responses. Metabolites of compound 2 formed in vitro by human liver microzones were characterized using high resolution tandem mass spectrometry. In sum, the in vivo activity of these compounds appears to be diminished by low bioavailability, but this experimental approach indicates the importance of systematic biomarker investigation.

The structure-activity relationships of A-ring-substituted aromathecin topoisomerase I inhibitors strongly support a camptothecin-like binding mode.

Aromathecins are inhibitors of human topoisomerase I (Top1). These compounds are composites of several heteroaromatic systems, namely the camptothecins and indenoisoquinolines, and they possess notable Top1 inhibition and cytotoxicity when substituted at position 14. The SAR of these compounds overlaps with indenoisoquinolines, suggesting that they may intercalate into the Top1-DNA complex similarly. Nonetheless, the proposed binding mode for aromathecins is purely hypothetical, as an X-ray structure is unavailable. In the present communication, we have synthesized eight novel series of A-ring-substituted (positions 1-3) aromathecins, through a simple, modular route, as part of a comprehensive SAR study. Certain groups (such as 2,3-ethylenedioxy) moderately improve Top1 inhibition, and, often, antiproliferative activity, whereas other groups (2,3-dimethoxy and 3-substituents) attenuate bioactivity. Strikingly, these trends are very similar to those previously observed for the A-ring of camptothecins, and this considerable SAR overlap lends further support (in the absence of crystallographic data) to the hypothesis that aromathecins bind in the Top1 cleavage complex as interfacial inhibitors in a 'camptothecin-like' pose.

Activity of indenoisoquinolines against African trypanosomes.

African trypanosomiasis (sleeping sickness), caused by protozoan Trypanosoma brucei species, is a debilitating disease that is lethal if untreated. Available drugs are antiquated, toxic, and compromised by emerging resistance. The indenoisoquinolines are a class of noncamptothecin topoisomerase IB poisons that are under development as anticancer agents. We tested a variety of indenoisoquinolines for their ability to kill T. brucei. Indenoisoquinolines proved trypanocidal at submicromolar concentrations in vitro. Structure-activity analysis yielded motifs that enhanced potency, including alkylamino substitutions on N-6, methoxy groups on C-2 and C-3, and a methylenedioxy bridge between C-8 and C-9. Detailed analysis of eight water-soluble indenoisoquinolines demonstrated that in trypanosomes the compounds inhibited DNA synthesis and acted as topoisomerase poisons. Testing these compounds on L1210 mouse leukemia cells revealed that all eight were more effective against trypanosomes than against mammalian cells. In preliminary in vivo experiments one compound delayed parasitemia and extended survival in mice subjected to a lethal trypanosome challenge. The indenoisoquinolines provide a promising lead for the development of drugs against sleeping sickness.

Design, synthesis, and biological evaluation of 14-substituted aromathecins as topoisomerase I inhibitors.

The aromathecin or "rosettacin" class of topoisomerase I (top1) inhibitors is effectively a "composite" of the natural products camptothecin and luotonin A and the synthetic indenoisoquinolines. The aromathecins have aroused considerable interest following the isolation and total synthesis of 22-hydroxyacuminatine, a rare cytotoxic natural product containing the 12 H-5,11a-diazadibenzo[ b, h]fluoren-11-one system. We have developed two novel syntheses of this system and prepared a series of 14-substituted aromathecins as novel antiproliferative topoisomerase I poisons. These inhibitors are proposed to act via an intercalation and "poisoning" mechanism identical to camptothecin and the indenoisoquinolines. Many of these compounds possess greater antiproliferative activity and anti-top1 activity than the parent unsubstituted compound (rosettacin) and previously synthesized aromathecins, as well as greater top1 inhibitory activity than 22-hydroxyacuminatine. In addition to potentially aiding solubility and localization to the DNA-enzyme complex, nitrogenous substituents located at the 14-position of the aromathecin system have been proposed to project into the major groove of the top1-DNA complex and hydrogen-bond to major-groove amino acids, thereby stabilizing the ternary complex.

Novel indenoisoquinolines NSC 725776 and NSC 724998 produce persistent topoisomerase I cleavage complexes and overcome multidrug resistance.

Camptothecin (CPT) derivatives are effective anticancer drugs, especially against solid tumors. As CPTs are chemically unstable and have clinical limitations, we have synthesized indenoisoquinolines as novel topoisomerase I (Top1) inhibitors. We presently report two indenoisoquinoline derivatives, NSC 725776 and NSC 724998, which have been selected for therapeutic development. Both are potent Top1 inhibitors and induce Top1 cleavage at unique genomic positions compared with CPT. Consistent with Top1 poisoning, protein-linked DNA breaks were detected in cells treated with NSC 725776 and NSC 724998 at nanomolar concentrations. Those drug-induced protein-linked DNA breaks persisted longer after drug removal than those produced by CPT. Studies in human cells in culture show that NSC 725776 and NSC 724998 exert antiproliferative activity at submicromolar concentrations. Furthermore, NSC 725776 and NSC 724998 show cross-resistance in cells deficient or silenced for Top1, which is consistent with their selective Top1 targeting. Similar to other known Top1 inhibitors, NSC 725776-treated and NSC 724998-treated cells show an arrest of cell cycle progression in both S and G(2)-M and a dependence on functional p53 for their cytotoxicity. Dose-dependent gamma-H2AX foci formation was readily observed in cells treated with NSC 725776 and NSC 724998. These gamma-H2AX foci were detectable at pharmacologically relevant doses for up to 24 h and thus could be used as biomarkers for clinical trials (phase 0).

Optimization of the indenone ring of indenoisoquinoline topoisomerase I inhibitors.

Two series of indenoisoquinoline topoisomerase I inhibitors have been prepared to investigate optimal substituents on the indenone ring at the 9-position. The more exhaustive series was prepared using a nitrated isoquinoline ring that has been previously demonstrated to enhance biological activity. After preliminary biological evaluation, a more focused series of inhibitors was prepared utilizing a 2,3-dimethoxy-substituted isoquinoline ring. The results of the two series indicate the existence of superior functional groups such as methoxy, fluorine, and cyano for the indenoisoquinoline 9-position. Interestingly, these functional groups coincide with established structure-activity relationships for the 11-position of camptothecin.

Nitrated indenoisoquinolines as topoisomerase I inhibitors: a systematic study and optimization.

The biological activity of indenoisoquinoline topoisomerase I (Top1) inhibitors can be greatly enhanced depending on the choice of substituents on the aromatic rings and lactam side chain. Previously, it was discovered that a 3-nitro group and a 9-methoxy group afforded enhanced biological activity. In the present investigation, indenoisoquinoline analogues were systematically prepared using combinations of nitro groups, methoxy groups, and hydrogen atoms in an effort to understand the contribution of each group toward cytotoxicity and Top1 inhibition. Analysis of the biological results suggests that the nitro group is important for Top1 inhibition and the methoxy group improves cytotoxicity. In addition, previously identified structure-activity relationships were utilized to select favorable lactam side chain functionalities for incorporation on the aromatic skeleton of analogues in this study. As a result, this investigation has provided optimal Top1 inhibitors equipotent to camptothecin that demonstrate low nanomolar cytotoxicities toward cancer cells.