School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand.
School of Biological Sciences, Victoria University of Wellington, Wellington 6012, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington 6012, New Zealand; Michael Smith Laboratories, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada.
Biochem Pharmacol. 2018 Dec;158:192-200. doi: 10.1016/j.bcp.2018.10.020. Epub 2018 Oct 21.
Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses and bacteria to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to the prodrug. Nitroreductases, able to activate a range of promising nitroaromatic prodrugs to genotoxic metabolites, are of great interest for GDEPT. The bystander effect (cell-to-cell transfer of activated prodrug metabolites) has been quantified for some nitroaromatic prodrugs in mixed multilayer human cell cultures, however while these provide a good model for viral DEPT (VDEPT) they do not inform on the ability of these prodrug metabolites to exit bacterial vectors (relevant to bacterial-DEPT (BDEPT)). To investigate this we grew two Escherichia coli strains in co-culture; an activator strain expressing the nitroreductase E. coli NfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activator to the recipient cells. We used this to investigate five clinically relevant prodrugs: metronidazole, CB1954, nitro-CBI-DEI, and two dinitrobenzamide mustard prodrug analogues, PR-104A and SN27686. Consistent with the bystander efficiencies previously measured in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. However, in contrast with observations in human cell multilayers, the nitrogen mustard prodrug metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, we further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products, consistent with their relative bystander efficiencies in human cell culture. Overall, our data suggest that prodrugs may differ in their suitability for VDEPT versus BDEPT applications and emphasise the importance of evaluating an enzyme-prodrug partnership in an appropriate context for the intended vector.
基因导向酶前药治疗（GDEPT）利用肿瘤趋向性载体，包括病毒和细菌，将基因编码的前药转化酶递送到肿瘤环境中，从而使肿瘤对前药敏感。硝基还原酶能够激活一系列有前途的硝基芳香族前药，将其转化为遗传毒性代谢物，因此对于 GDEPT 非常感兴趣。一些硝基芳香族前药在混合多层人细胞培养物中的旁观者效应（激活的前药代谢物在细胞间的转移）已经被定量，然而，虽然这些为病毒 DEPT（VDEPT）提供了一个很好的模型，但它们并不能说明这些前药代谢物离开细菌载体的能力（与细菌 DEPT（BDEPT）相关）。为了研究这一点，我们在共培养中生长了两种大肠杆菌菌株；一种表达硝基还原酶大肠杆菌 NfsA 的激活菌株和一种含有 SOS-GFP DNA 损伤反应基因构建体的受体菌株。在这个系统中，只有在前药代谢物从激活剂转移到受体细胞后，才能诱导 GFP 的表达。我们用它来研究五种临床相关的前药：甲硝唑、CB1954、硝基-CBI-DEI 和两种二硝基苯甲酰胺芥前药类似物 PR-104A 和 SN27686。与先前在人细胞多层中测量的旁观者效率一致，还原甲硝唑表现出很少的细菌细胞间转移，而硝基-CBI-DEI 在还原后从激活剂非常有效地传递到受体细胞。然而，与在人细胞多层中的观察结果相反，氮芥前药代谢物在两种细菌菌株之间不能有效地传递，而还原 CB1954 则有效地传递。使用对 CB1954 的 2-和 4-硝基取代基表现出不同偏向性的硝基还原酶，我们进一步表明，2-硝基还原产物表现出比 4-硝基还原产物更高水平的细菌细胞间转移，这与它们在人细胞培养物中的旁观者效率相对应。总的来说，我们的数据表明，前药可能在适合 VDEPT 与 BDEPT 应用方面存在差异，并强调在适当的载体背景下评估酶-前药伙伴关系的重要性。
