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.

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.

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.

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.

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.

Investigation of the lactam side chain length necessary for optimal indenoisoquinoline topoisomerase I inhibition and cytotoxicity in human cancer cell cultures.

Indenoisoquinolines with lactam substituents such as ethylamino, propylamino, and butylamino have previously demonstrated potent biological activity, but an optimal length has never been established. In the present study, a series of simplified indenoisoquinoline analogues possessing a linker spacing of 0-12 carbon atoms between the lactam nitrogen and the terminal amino group have been prepared, determining that 2-4-atom lengths are optimal for topoisomerase I inhibition and cytotoxicity. Using these lengths, analogues were prepared with the amino group and portions of the linker replaced by a pyridine ring. A three-carbon spacer within the pyridine series still demonstrated potent topoisomerase I inhibition.

A systematic study of nitrated indenoisoquinolines reveals a potent topoisomerase I inhibitor.

The biological activity of indenoisoquinoline topoisomerase I inhibitors is significantly enhanced by nitration of the isoquinoline ring. In the present study, nitrated analogues were synthesized with the indenone ring substituted with methoxy groups to further explore a previously identified structure-activity relationship between the nitrated isoquinoline ring and a methylenedioxy-substituted indenone ring. The results indicate that a single methoxy group at the 9-position of an indenoisoquinoline affords superior biological activity. Hypothetical binding models have been developed to rationalize these results, and they indicate that pi-stacking between the indenoisoquinolines and the DNA base pairs, as visualized by electrostatic complementarity, is important for the intercalation and biological activity of the indenoisoquinoline analogues. Collectively, the analysis of methoxy groups on the indenone ring also illustrates a strict steric requirement for substituents extending toward the nonscissile DNA backbone and emphasizes a need for planarity to afford potent biological activity.

Synthesis and evaluation of indenoisoquinoline topoisomerase I inhibitors substituted with nitrogen heterocycles.

In connection with an ongoing investigation of indenoisoquinoline topoisomerase I (Top1) inhibitors as potential therapeutic agents, the pharmacophore possessing di(methoxy) and methylenedioxy substituents was held constant, and new derivatives were synthesized with nitrogen heterocycles appended to the lactam side chain. Compounds were evaluated for Top1 inhibition and for cytotoxicity in the National Cancer Institute's human cancer cell screen. Some of the more potent derivatives were also screened for in vivo activity in a hollow fiber assay. The results of these studies indicate that lactam substituents possessing nitrogen heterocycles can provide highly cytotoxic compounds with potent Top1 inhibition. Molecular modeling of these compounds in complex with DNA and Top1 suggests that some of the lactam substituents are capable of interacting with the DNA base pairs above and below the site of intercalation and/or with Top1 amino acid residues, resulting in increased biological activity.

Synthesis and biological evaluation of bisindenoisoquinolines as topoisomerase I inhibitors.

The indenoisoquinolines represent a class of non-camptothecin topoisomerase I (Top1) inhibitors that exert cytotoxicity by trapping the covalent complex formed between DNA and Top1 during relaxation of DNA supercoils. As an ongoing evaluation of Top1 inhibition and anticancer activity, indenoisoquinolines were linked via their lactam side chains to provide polyamines end-capped with intercalating motifs. The resulting bisindenoisoquinolines were evaluated for cytotoxicity in the National Cancer Institute's human cancer cell screen and for Top1 inhibition. Preliminary findings suggested that the 2-3-2 and 3-3-3 linkers, referring to the number of carbons between nitrogen atoms, were optimal for both potent Top1 inhibition and cytotoxicity. Using optimized linkers, bisindenoisoquinolines were synthesized with nitro and methoxy substituents on the aromatic rings. The biological results for substituted compounds revealed a disagreement between the structure-activity relationships of monomeric indenoisoquinolines and bisindenoisoquinolines as Top1 inhibitors, but cytotoxicity was maintained for both series of compounds.

Solid-phase compound library synthesis in drug design and development.

This article is a subjective review of the literature between December 2000 and January 2002 related to the solid-phase synthesis of compound libraries for drug discovery and development. Examples of libraries yielding active compounds across a range of biological targets are presented, together with synthetic details where this is deemed of sufficient note. Background to the biological target and any structure-activity relationship, which can be discerned within members of a library series, is also discussed. A brief overview of new developments in solid-phase technology that is likely to impact upon drug discovery is also included.

Synthesis and anticancer activity of simplified indenoisoquinoline topoisomerase I inhibitors lacking substituents on the aromatic rings.

The indenoisoquinolines are a class of cytotoxic topoisomerase I inhibitors that offer certain advantages over the camptothecins, including the greater stabilities of the compounds themselves, as well as the greater stabilities of their drug-enzyme-DNA cleavage complexes. To investigate the possible biological roles of the di(methoxy) and methylenedioxy substituents present on the aromatic rings of the previously synthesized indenoisoquinoline topoisomerase I inhibitors, a series of compounds lacking these substituents was synthesized and tested for both cytotoxicity in cancer cell cultures and for enzyme inhibitory activity. The results indicate that the aromatic substituents make a small, but consistently observable contribution to the biological activity. Molecular models derived for the binding of the unsubstituted indenoisoquinolines in ternary complex with DNA and topoisomerase I indicate that the substituents on the lactam nitrogen project out of the major groove, and the carbonyl group is directed out of the minor groove, where it is involved in a hydrogen bonding interaction with the side chain guanidine group of Arg364. The DNA cleavage patterns observed in the presence of topoisomerase I and various indenoisoquinolines were similar, although significant differences were detected. There were also variations in the DNA cleavage pattern seen with camptothecin vs the indenoisoquinolines, which indicates that these two classes of topoisomerase I inhibitors are likely to target the cancer cell genome differently, resulting in different spectra of anticancer activity. The most cytotoxic of the presently synthesized indenoisoquinolines has a 4-amino-n-butyl group on the lactam nitrogen.

Blindness and rectus muscle damage following spinal surgery.

PURPOSE: To report a rare case of blindness and rectus muscle damage from ocular compression after spinal surgery. DESIGN: Observational case report. METHODS: A 16-year-old girl underwent scoliosis surgery in the prone position. After surgery, she complained of visual loss in the right eye. Examination showed no perception of light, periorbital bruising, corneal haze, fixed dilated pupil, and loss of adduction beyond the midline. RESULTS: Orbital computed tomography scan showed a swollen medial rectus with no other orbital pathology. The muscle swelling can be explained by ischemia from compression of the globe against the medial wall of the orbit. Ocular compression also caused retinal and optic nerve ischemia, resulting in loss of vision. Medial rectus function recovered with time, but the patient continues to have no light perception. CONCLUSIONS: Blindness after spinal surgery is a rare but devastating complication, and all precautions to avoid ocular compression must be taken.

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.