Synthesis and evaluation of aryliden- and hetarylidenfuranone derivatives of usnic acid as highly potent Tdp1 inhibitors

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a repair enzyme for stalled DNA-topoisomerase 1 (Top 1) cleavage complexes and other 3′-end DNA lesions. Tdp1 is a promising target for anticancer therapy, since it can repair DNA lesions caused by Top1 inhibitors leading to drug resistance. Hence, Tdp1 inhibition should result in synergistic effect with Top1 inhibitors. Twenty nine derivatives of (+)-usnic acid were tested for in vitro Tdp1 inhibitory activity using a fluorescent-based assay. Excellent activity was obtained, with derivative 6m demonstrating the lowest IC₅₀ value of 25?nM. The established efficacy was verified using a gel-based assay, which gave close results to that of the fluorescent assay. In addition, molecular modeling in the Tdp1 substrate binding pocket suggested plausible binding modes for the active analogues. The synergistic effect of the Tdp1 inhibitors with topotecan, a Top1 poison in clinical use, was tested in two human cell lines, A-549 and HEK-293. Compounds 6k and 6x gave very promising results. In particular, 6x has a low cytotoxicity and an IC₅₀ value of 63?nM, making it a valuable lead compound for the development of potent Tdp1 inhibitors for clinical use.     

Micro- and macromimetics: Condensation of amino acid derivatives with dienophiles

Summary Heterocycles (pyridines and pyrroles) obtained from amino acid derivatives and unsaturated compounds can be considered as peptidomimetics. These compounds contain amino acid radicals and modified, nonhydrolysable peptide bonds (micromimetics). The polyfunctionality of pyridoxine (one of the most available pyridines, derived from N-formyl -alanine amide/ester) offers the possibility for use in the synthesis of multiple peptides (MPs). In view of the fact that MPs have certain structures and molecular weights and also owing to the proximity of several peptide chains, they may be considered as models of proteins and even of cell surfaces (macromimetics). Dendritic compounds — multiple spherical peptides — most closely imitate globular proteins. The potential for macromimetics to model the various functions of biopolymers is investigated here.     

Organization and chromosomal localization of a B1-like containing repeat of Microtus subarvalis

A repetitive DNA sequence, MS2, was isolated from EcoRI-digested genomic DNA of the vole, Microtus subarvalis. The fragment was cloned and sequenced. Sequence analysis of this 1194-bp fragment revealed a 156-bp region demonstrating a 55% homology with the mouse B1 repeat. The remaining MS2 sequence shows no significant homology with other known GenBank sequences. The results of in situ hybridization of MS2 on vole metaphase chromosomes indicate the fragment is confined to heterochromatin blocks of the sex chromosomes in all but one species (M. arvalis). Distribution of MS2 sequences provides evidence for heterogeneity of the giant heterochromatin blocks of the XY Chromosomes (Chrs) in voles, for the unique cluster-like localization of MS2 within these blocks. Received: 10 October 1995 / Accepted: 30 March 1996     

Mouse Spermatozoa in High Concentrations of Glycerol: Chemical Toxicity vs Osmotic Shock at Normal and Reduced Oxygen Concentrations

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilities or irreproducibility. We have identified several likely underlying problems. One is that published studies have used concentrations of the cryoprotectant glycerol that are substantially lower (0.3 M) than the ∼1 M concentrations that are optimal for most cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We have identified two contributors to damage from higher concentrations of glycerol, namely, chemical toxicity proportional to concentration and exposure time and osmotic damage arising from too rapid an addition and removal of the glycerol. When toxicity is minimized by restricting the exposure time to 1 or 5 min and osmotic shock is minimized by adding and removing the glycerol stepwise, relatively high percentages of the sperm survive contact with 0.8 M glycerol. Free-radical damage in mouse sperm is known to be proportional to the oxygen concentration. We have determined the consequences of reducing the oxygen to <3% of atmospheric by the use of a bacterial membrane preparation, Oxyrase. Oxyrase reduced damage from centrifugation and substantially reduced damage from osmotic shock; however, it did not significantly reduce glycerol toxicity.     

Lipophilic Metabolites from Five‐Needle Pines,Pinus armandii and Pinus kwangtungensis,Exhibiting Antibacterial Activity

Lipophilic extractive metabolites from needles and defoliated twigs of Pinus armandii and P. kwangtungensis were studied by GC/MS. Needles of P. armandii contained predominantly 15‐O‐functionalized labdane type acids (anticopalic acid), fatty acids, nonacosan‐10‐ol, sterols, nonacosan‐10‐ol and sterol saponifiable esters, and acylglycerols, while P. kwangtungensis needles contained no anticopalic acid, but more trinorlabdane (14,15,16‐trinor‐8(17)‐labdene‐13,19‐dioic acid) and other labdane type acids, nonacosan‐10‐ol and its saponifiable esters. The major compounds in the P. armandii defoliated twig extract were abietane and isopimarane type acids, fatty acids, sterols, labdanoids (cis‐abienol), cembranoids (isocembrol and 4‐epi‐isocembrol), saponifiable sterol esters, and acylglycerols. The same extract of P. kwangtungensis contained larger quantities of fatty acids, caryophyllene oxide, serratanoids, sterols, saponifiable sterol esters, and acylglycerols, but lesser amounts of abietane and isopimarane type acids, cis‐abienol, and lacked cembranoids. Both twig and needle extracts of P. armandii and P. kwangtungensis, as well as the extracts’ fractions, significantly inhibited the growth of Gram‐negative bacteria Serratia marcescens with MIC of 0.1 mg ml^?1, while in most cases they slightly stimulated the growth of Gram‐positive bacteria Bacillus subtilis at the same concentrations. Thus, lipophilic extractive compounds from the needles and defoliated twigs of both pines are prospective for the development of antiseptics against Gram‐negative bacteria.