An RNA model system consisting of an oligomer binding to a 4-nt overhang at the 5' end ofa hairpin stem provides thermodynamic parameters for helix-helix interfaces. In a sequence-dependent manner, oligomers bind up to 1000-fold more tightly adjacent to the hairpin stem than predicted for binding to a free tetramer at 370C. For reaction (19, 20). Synthetic details will be provided elsewhere.Measurement of Thermodynamic Parameters. The buffer for melting experiments was 1 M NaCl/10 mM sodium cacodylate/0.5 mM EDTA. Absorbance versus temperature curves were measured at 280 nm with a heating rate of 10C/min. These curves were analyzed by fitting to a two-state model with sloping base lines through use of a nonlinear least-squares program (21).Predictions of Secondary Structure. Sets of 20 secondary structures were generated with the program of Zuker (12), kcal/mol, and internal loops of three were given a AG'7 for loop closure of 5.1 + 0.3 = 5.4 kcal/mol (23). (iii) Stacking and pairing of terminal mismatches were included in calculating free energies of internal loops in the following manner. Single mismatches were given AG7 = 0.8 kcal/mol. For larger internal loops, terminal GA, UU, and other mismatches adjacent to COG pairs were given favorable free energy increments of -2.7, -2.5, and -1.5 kcal/mol, respectively (24). These mismatches adjacent to A-U pairs were given AG' values of -2. 9218The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Alkylating agents which are activated by glutathion-S-transferases (GSTs) have been designed and synthesized. The model compound gamma-glutamyl-alpha-amino-beta-[(2-ethyl N,N,N',N'-tetraethylphosphorodiamidate) sulfonyl]propionylglycine (1) and the nitrogen mustards gamma-glutamyl-alpha- amino-beta-[[2-ethyl N,N,N',N'-tetrakis (2-chloroethyl)phosphorodiamidate] sulfonyl]propionylglycine (2) and gamma-glutamyl-alpha-amino-beta-[[2-ethyl-N,N,N',N'-tetrakis(2- chloroethyl)phosphorodiamidate]sulfonyl]-propionyl-(R)-(-)-phenylg lycine (3) were prepared via multistep chemical synthesis. The compounds were tested with recombinant human A1-1, M1a-1a and P1-1 GSTs. HPLC studies showed that the compounds were differentially and catalytically cleaved by biologically relevant concentrations of the GSTs. Mass spectral studies of the cleavage mixture of 2 showed that M1a-1a GST liberated the cytotoxic phosphate moiety needed for efficacy as an alkylating agent. Cell culture studies with MCF-7 breast cancer cells showed that 1 was not toxic at 200 microM, while 2 and 3 showed IC50S of 40.6 and 37.5 microM, respectively, for the same cell line. MCF-7 cells transfected to overexpress P1-1 GST showed enhanced sensitivity with 2 and 3, with IC50S of 20.9 and 9.5 microM, respectively. This result correlates well with the rates of cleavage of 2 and 3 by P1-1 GST observed in vitro and demonstrates that higher levels of cellular P1-1 GST will give increased sensitivity to these drugs.
A method is presented for the incorporation of nonnatural amino acids into proteins during in vitro cell-free translation. A combination of chemical synthesis and run-off transcription was employed to prepare a semisynthetic, nonhypermodified tRNA(Gly) nonsense suppressor acylated with L-3-[125I]iodotyrosine. The presence of this synthetic tRNA during in vitro translation of mRNA containing a nonsense suppression site (e.g., a UAG termination codon) results in the incorporation of the nonnatural amino acid L-3-iodotyrosine into the polypeptide exclusively at the position corresponding to that site. Incorporation of the nonnatural amino acid L-3-[125I]iodotyrosine into the model polypeptide was assessed by quantitative and unambiguous determination of suppression efficiency, read-through, and site specificity of incorporation. Minor modifications of the method employed in this initial experiment also allow the rapid analysis of unlabeled acylated tRNA analogues. Under optimum conditions, the unlabeled amino acid L-3-iodotyrosine was found to be incorporated with a suppression efficiency of 65%. Other nonnatural residues, including N-methylphenylalanine, D-phenylalanine, and phenyllactic acid, were tested in the assay under these same conditions. Suppression efficiencies for this series ranged from 0 to 72% depending on the structure of the residue incorporated. Several other aspects of this methodology, such as tRNA structure and context effects, are briefly discussed.
In search of compounds with improved specificity for targeting the important cancer-associated P1-1 glutathione S-transferase (GST) isozyme, new analogs 4 and 5 of the previously reported glutathione S-transferase (GST)-activated latent alkylating agent gamma-glutamyl-alpha-amino-beta-[[[2-[[bis[bis(2-chloroethyl)amino]ph osp horyl]oxy]ethyl]sulfonyl]propionyl]-(R)-(-)-phenylglycine (3) have been designed, synthesized, and evaluated. One of the diastereomers of 4 exhibited good selectivity for GST P1-1. The tetrabromo analog 5 of the tetrachloro compound 3 maintained its specificity and was found to be more readily activated by GSTs than 3. The GST activation concept was further broadened through design, synthesis, and evaluation of a novel latent urethane mustard 8 and its diethyl ester 9. Interestingly, 8 showed very good specificity for P1-1 GST. Cell culture studies were carried out on 4, 5, 8, and 9 using cell lines engineered to have varying levels of GST P1-1 isozyme. New analogs 4 and 5 exhibited increased toxicity to cell lines with overexpressed GST P1-1 isozyme. The urethane mustard 8 and its diethyl ester 9 were found to be not as toxic. However, they too exhibited more toxicity to a cell line engineered to have elevated P1-1 levels, which was in agreement with the observed in vitro specificity of 8 for P1-1 GST isozyme. Mechanistic studies on alkaline as well as enzyme-catalyzed decomposition of latent mustard 3 provided experimental proof for the hypothesis that 3 breaks down into an active phosphoramidate mustard and a reactive vinyl sulfone. The alkylating nature of the decomposition products was further demonstrated by trapping those transient species as relatively stable diethyldithiocarbamic acid adducts. These results substantially extend previous efforts to develop drugs targeting GST and provide a paradigm for development of other latent drugs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.