Abstract
New isopropylated fused azaisocytosine-containing congeners (I–VI) have previously been reported as promising anticancer drug candidates, so further research on these molecules in the preclinical development phase is fully justified and necessary. For this reason, in the present paper, we assess the toxicity/safety profiles of all the compounds using Danio rerio and red blood cell models, and examine the effect of the most selective congeners on the activation of apoptotic caspases in cancer and normal cells. In order to evaluate the effect of each molecule on the development of zebrafish embryos/larvae and to select the safest compounds for further study, various phenotypic parameters (i.e., mortality, hatchability, heart rate, heart oedema, yolk sac utilization, swim bladder development and body shape) were observed, and the half maximal lethal concentration, the maximal non-lethal concentration and no observed adverse effect concentration for each compound were established. The effect of all the isopropylated molecules was compared to that of an anticancer agent pemetrexed. The lipophilicity-dependent structure–toxicity correlations were also determined. To establish the possible interaction of the compounds with red blood cells, an ex vivo hemolysis test was performed. It was shown that almost all of the investigated isopropylated congeners have no adverse phenotypic effect on zebrafish development during five-day exposure at concentrations up to 50 μM (I–III) or up to 20 μM (IV–V), and that they are less toxic for embryos/larvae than pemetrexed, demonstrating their safety. At the same time, all the molecules did not adversely affect the red blood cells, which confirms their very good hemocompatibility. Moreover, they proved to be activators of apoptotic caspases, as they increased caspase-3, -7 and -9 levels in human breast carcinoma cells. The conducted research allows us to select—from among the anticancer active drug candidates—compounds that are safe for developing zebrafish and red blood cells, suitable for further in vivo pharmacological tests.
Highlights
We reported in our earlier paper [1] a novel class of isopropylated fused azaisocytosinecontaining congeners (I–VI, Figure 1), which was synthesized in our laboratory as a result of the fruitful isosteric replacement
As the development of highly safe drugs is desirable, each newly synthesized potential drug candidate should be carefully assessed for its toxicity/safety in an animal model already at an early stage of its preclinical development, before entering the clinical trials [4,5,6]
To assess the adverse effects of test compounds on zebrafish development, we studied observable phenotypic parameters—such as the mortality, ability to hatch, heart rate, heart oedema, yolk sac utilization, swim bladder development and body shape—in embryos/larvae that were exposed to the compounds/pemetrexed at various concentrations
Summary
We reported in our earlier paper [1] a novel class of isopropylated fused azaisocytosinecontaining congeners (I–VI, Figure 1), which was synthesized in our laboratory as a result of the fruitful isosteric replacement. In this way, promising drug candidates were obtained which—being isosteres of endogenic nucleobases—can act as irreversible inactivators of specific enzymes (antimetabolites). One of the appropriate research models is the recently very popular organism—the zebrafish (Danio rerio). This small fish is an attractive alternative to research on other vertebrates, such as mice and rats. Iat bissohrobpetdhethmatothleecmuloesst sferloemctivtehemwoleactuelres[4o,f5t,h9i,s10cl]a.sAs wllitllhfeinsde faepaptluicraetisonmianke Danio rerio a suitable cancermchoedmeolthtoerasptuy dinythme afuntuyrea.spects of toxicity in the preclinical phase of drug development
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