Pseudomonas aeruginosa NfsB and nitro-CBI-DEI--a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy.

BACKGROUND: The nitro-chloromethylbenzindoline prodrug nitro-CBI-DEI appears a promising candidate for the anti-cancer strategy gene-directed enzyme prodrug therapy, based on its ability to be converted to a highly cytotoxic cell-permeable derivative by the nitroreductase NfsB from Escherichia coli. However, relative to some other nitroaromatic prodrugs, nitro-CBI-DEI is a poor substrate for E. coli NfsB. To address this limitation we evaluated other nitroreductase candidates from E. coli and Pseudomonas aeruginosa. FINDINGS: Initial screens of candidate genes in the E. coli reporter strain SOS-R2 identified two additional nitroreductases, E. coli NfsA and P. aeruginosa NfsB, as being more effective activators of nitro-CBI-DEI than E. coli NfsB. In monolayer cytotoxicity assays, human colon carcinoma (HCT-116) cells transfected with P. aeruginosa NfsB were >4.5-fold more sensitive to nitro-CBI-DEI than cells expressing either E. coli enzyme, and 23.5-fold more sensitive than untransfected HCT-116. In three dimensional mixed cell cultures, not only were the P. aeruginosa NfsB expressing cells 540-fold more sensitive to nitro-CBI-DEI than pure cultures of untransfected HCT-116, the activated drug that they generated also displayed an unprecedented local bystander effect. CONCLUSION: We posit that the discrepancy in the fold-sensitivity to nitro-CBI-DEI between the two and three dimensional cytotoxicity assays stems from loss of activated drug into the media in the monolayer cultures. This emphasises the importance of evaluating high-bystander GDEPT prodrugs in three dimensional models. The high cytotoxicity and bystander effect exhibited by the NfsB_Pa/nitro-CBI-DEI combination suggest that further preclinical development of this GDEPT pairing is warranted.

Vital functions of corticotropin-releasing factor (CRF) pathways in maintenance and regulation of energy homeostasis.

Regulation of energy homeostasis is a vital function of the CNS requiring adaptive responses to maintain and support life after stress perturbations. The mechanisms whereby these processes occur are, however, only partially understood. A major determinate of these responses is corticotropin-releasing factor (CRF). Receptors for CRF, CRFR1 and CRFR2, have been hypothesized to play distinct roles in the alterations necessary for homeostatic maintenance. The function of CRFR2, in particular, has remained elusive despite its presence in both the CNS and periphery. In this work, we have used complimentary gene deletion and pharmacological approaches to elucidate the crucial role CRFR2 plays in the regulation of regional tissue thermogenesis and adaptive physiology. Analyses of interscapular brown adipose tissue (IBAT) thermogenesis by thermal signature analysis and the concordant biochemical changes in key sympathetic components in mice deficient for CRFR2 revealed significantly elevated basal IBAT thermogenesis and prolonged adrenergic responsivity of IBAT in older mice. Measurement of metabolic rates by indirect calorimetry after chronic high-fat diet challenge and treatment with the CRFR1 antagonist NBI-27914 revealed a decreased respiratory exchange ratio of these mice that was normalized with NBI-27914. Further, as a definitive measure for physiological pathology, mice examined in a behavioral model of differential temperature selection showed a predilection for warmer external temperatures, supporting a loss of body heat in these mice. These studies provide physiological, biochemical, and behavioral evidence for the critical participation of CRF pathways in the maintenance and adaptive responses necessary for regulation of energy homeostasis.