Darshan Makhey

Selective cytotoxicity of topoisomerase-directed protoberberines against glioblastoma cells.

Protoberberines are a new class of organic cations that are dual poisons of topoisomerases I and II. Certain protoberberines exhibit greater in vitro cytotoxicity against cell lines derived from solid tumors than from leukemias. Using a group of seventeen different protoberberine analogs, the structural basis for selective cytotoxicity toward sensitive SF-268 glioblastoma cells as compared with resistant RPMI 8402 lymphoblast cells was explored. The selective cytotoxicity is associated with the presence of an imminium ion and other structural features of protoberberines, and is not shared by drugs such as camptothecin, doxorubicin, vinblastine, and etoposide, which are either equally or more cytotoxic against RPMI 8402 cells than SF-268 cells. The selective cytotoxicity of protoberberines against SF-268 over RPMI 8402 cells is not due to differences in topoisomerase levels or known drug efflux systems such as multidrug resistance (MDR1) and multidrug-resistance protein (MRP). Comparative in vitro studies of the accumulation of coralyne, a fluorescent protoberberine, into sensitive and resistant cells demonstrated a correlation between drug accumulation and selective cytotoxicity. Inhibitors of coralyne uptake included several protoberberine-related compounds. Of these, palmatine, a minimally cytotoxic protoberberine, both inhibited coralyne accumulation and reduced cytotoxicity against SF-268 cells, but not against RPMI 8402 cells. Despite the structural resemblance of protoberberines to catecholamines, our experiments using inhibitors and cells expressing biogenic amine uptake systems have ruled out the involvement of biogenic amine uptake1, uptake2, and vesicular monoamine transport systems. Uptake systems remaining as candidates, supported by preliminary data, include transport via vesicles derived from specialized membrane invaginations and selected carrier-mediated organic amine transport systems.

Substituted benzo[i]phenanthridines as mammalian topoisomerase-Targeting agents

Several benzo[c]phenanthridine and protoberberine alkaloids, such as nitidine and berberrubine, are known to induce DNA cleavage in the presence of either topoisomerase I or II. Structure-activity studies performed on various analogues related to benzo[c]phenanthridine and protoberberine alkaloids have provided insights into structural features that influence this topoisomerase-targeting activity. Modifications within the A-ring of benzo[c]phenanthridine and protoberberine alkaloids can significantly alter their ability to enhance the cleavable complex formation that occurs between DNA and topoisomerases. Select benzo[i]phenanthridines were synthesized as potential bioisosteres of nitidine and its analogues. In the present study, 2,3-methylenedioxy-8,9-dimethoxybenzo[i]phenanthridine, 2,3-methylenedioxy-8,9-dimethoxy-5-methylbenzo[i]phenanthridine, 2,3,8,9-tetramethoxybenzo[i]phenanthridine and 5-methyl-2,3,8,9-tetramethoxybenzo[i]phenanthridine were synthesized. These benzo[i]phenanthridine derivatives were evaluated for their ability to enhance cleavable complex formation in the presence of topoisomerases and DNA as well as for their cytotoxicity against the human lymphoblastoma cell line, RPMI8402. 2,3-Methylenedioxy-8,9-dimethoxybenzo[i]phenanthridine (4a) and its 5-methyl derivative (4b) are active as topoisomerase I-targeting agents. In contrast to nitidine, the presence of the 5-methyl substituent in the case of 4b is not associated with enhanced activity. Consistent with previous structure-activity studies on nitidine and protoberberine alkaloids, 2,3,8,9-teramethoxybenzo[i]phenanthridine, 5a, and its 5-methyl derivative, 5b, are inactive as topoisomerase I-targeting agents. These studies were extended to an evaluation of the relative pharmacological activities of 2,8,9-trimethoxybenzo[i]phenanthridine, 3,8,9-trimethoxybenzo[i]phenanthridine, and 2,3-methylenedioxy-8,9-methylenedioxybenzo[i]phenanthridine.

Substituted benz[a]acridines and benz[c]acridines as mammalian topoisomerase poisons.

Coralyne and several other synthetic benzo[a,g]quinolizium derivatives related to protoberberine alkaloids have exhibited activity as topoisomerase poisons. These compounds are characterized by the presence of a positively charged iminium group, which has been postulated to be associated with their pharmacological properties. The objective of the present study was to devise stable noncharged bioisosteres of these compounds. Several similarly substituted benz[a]acridine and benz[c]acridine derivatives were synthesized and their relative activity as topoisomerase poisons was determined. While the benz[c]acridine derivatives evaluated as part of this study were devoid of topoisomerase poisoning activity, several dihydrobenz[a]acridines were able to enhance DNA cleavage in the presence of topo I. In contrast to certain protoberberine derivatives that did exhibit activity as topo II poisons, none of the benz[a]acridines derivatives enhanced DNA cleavage in the presence of topo II. Among the benz[a]acridines studied, 5,6-dihydro-3,4-methylenedioxy-9,10-dimethoxybenz[a]acridine, 13e, was the most potent topo I poison, with comparable potency to coralyne. These data suggest that heterocyclic compounds structurally related to coralyne can exhibit potent topo I poisoning activity despite the absence of an iminium cation within their structure. In comparison to coralyne or other protoberberine derivatives, these benz[a]acridine derivatives possess distinctly different physicochemical properties and represent a novel series of topo I poisons.

EFFECTS OF TOPOISOMERASES INHIBITORS PROTOBERBERINE ON LEISHMANIA DONOVANI GROWTH, MACROPHAGE FUNCTION, AND INFECTION

DNA topoisomerases play a pivotal role in the regulation of cell division. Inhibition of Leishmania spp. topoisomerases represents an alternative to control parasite growth. Cancer research led to the development of several potent topoisomerase inhibitors such as topoisomerase I, topoisomerase II, or both (monobenzimidazole, terbenzimidazole, and protoberberine alkaloid-related compounds) that are effective antitumor agents. In the present study, we evaluated the efficacy of these compounds against Leishmania spp. growth in vitro. Some protoberberine compounds showed pronounced antileishmanial activity and were selected for further analysis in macrophages. These compounds did not affect macrophage viability and only slightly reduced macrophage nitric oxide generation in response to interferon-γ. Moreover, exposure of infected macrophages to these compounds significantly reduced parasite loads. Collectively, our data suggest that protoberberine-related compounds have powerful antileishmania action and that minor structural variations among them can substantially improve their activity to restrict Leishmania spp. infection in vitro.