Smart biomaterials have the ability to respond to changes in physiological parameters and exogenous stimuli and continue to impact many aspects of modern medicine. Smart materials can promote promising therapies and improve treatment of debilitating diseases. Here, we describe recent advances in the current state-of-the-art design and application of smart biomaterials in tissue engineering, drug delivery systems, medical devices, and immune engineering.
The disaccharide moiety is responsible for the tumor cell targeting properties of bleomycin (BLM). While the aglycon (deglycobleomycin) mediates DNA cleavage in much the same fashion as bleomycin, it exhibits diminished cytotoxicity in comparison to BLM. These findings suggested that BLM might be modular in nature, composed of tumor-seeking and tumoricidal domains. To explore this possibility, BLM analogues were prepared in which the disaccharide moiety was attached to deglycobleomycin at novel positions, namely, via the threonine moiety or C-terminal substituent. The analogues were compared with BLM and deglycoBLM for DNA cleavage, cancer cell uptake, and cytotoxic activity. BLM is more potent than deglycoBLM in supercoiled plasmid DNA relaxation, while the analogue having the disaccharide on threonine was less active than deglycoBLM and the analogue containing the C-terminal disaccharide was slightly more potent. While having unexceptional DNA cleavage potencies, both glycosylated analogues were more cytotoxic to cultured DU145 prostate cancer cells than deglycoBLM. Dye-labeled conjugates of the cytotoxic BLM aglycons were used in imaging experiments to determine the extent of cell uptake. The rank order of internalization efficiencies was the same as their order of cytotoxicities toward DU145 cells. These findings establish a role for the BLM disaccharide in tumor targeting/uptake and suggest that the disaccharide moiety may be capable of delivering other cytotoxins to cancer cells. While the mechanism responsible for uptake of the BLM disaccharide selectively by tumor cells has not yet been established, data are presented which suggest that the metabolic shift to glycolysis in cancer cells may provide the vehicle for selective internalization.
Two fluorescent amino acids, including the novel fluorescent species biphenyl-phenylalanine, have been incorporated into positions 17 and 115 of dihydrofolate reductase to enable a study of conformational changes associated with inhibitor binding. Unlike most studies involving fluorescently labeled proteins, the fluorophores were incorporated into the amino acid side chains, and both probes (biphenyl-phenylalanine (1) and L-(7-hydroxycoumarin-4-yl)ethylglycine (2)) were smaller than fluorophores typically used for such studies. The DHFR positions were chosen as potentially useful for FRET measurements based on their estimated separation (~17–18 Å), and expected change in distance along the reaction coordinate. Also of interest was the steric accessibility of the two sites: Glu17 is on the surface of DHFR, while Ile115 is within a folded region of the protein. Modified DHFR I (1 at position 17; 2 at position 115) and DHFR II (2 at position 17; 1 at position 115) were both catalytically competent. However, DHFR II, containing the potentially rotatable biphenyl-phenylalanine moiety at sterically encumbered position 115 was significantly more active than DHFR I. Irradiation of the modified DHFRs at 280 nm effected excitation of biphenyl-phenylalanine (1), energy transfer to coumarin 2 and emission at 450 nm. However, energy transfer was substantially more efficient for DHFR II. The effect of inhibitor binding was also measured. Trimethoprim mediated concentration dependent diminution of the emission observed at 450 nm for DHFR II, but not for DHFR I. These findings demonstrate that amino acids containing small fluorophores can be introduced into DHFR with minimal disruption of function, and in a fashion that enables sensitive monitoring of changes in DHFR conformation.
Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of Escherichia coli dihydrofolate reductase (ecDHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (A) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, D) at position 17. Further, 6-CNTrp (A) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving A as a fluorescence donor and benzo[g]quinazoline-2,4-(1H,3H)-dione 2'-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2'-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the BCL2 i-motif DNA (labeled with Cf). Thus, the non-natural amino acid could be used as a FRET partner for studying protein-nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (A) as a fluorescence donor for the study of protein conformational events.
Recently, we reported that both bleomycin (BLM) and its disaccharide, conjugated to the cyanine dye Cy5**, bound selectively to cancer cells. Thus, the disaccharide moiety alone recapitulates the tumor cell targeting properties of BLM. Here, we demonstrate that the conjugate of the BLM carbamoylmannose moiety with Cy5** showed tumor cell selective binding and also enhanced cellular uptake in most cancer cell lines. The carbamoyl functionality was required for tumor cell targeting. A dye conjugate prepared from a trivalent cluster of carbamoylmannose exhibited levels of tumor cell binding and internalization significantly greater than those of the simple carbamoylmannose–dye conjugate, consistent with a possible multivalent receptor.
We have shown previously that the bleomycin (BLM) carbohydrate moiety can recapitulate the tumor cell targeting effects of the entire BLM molecule, that BLM itself is modular in nature consisting of a DNA-cleaving aglycone which is delivered selectively to the interior of tumor cells by its carbohydrate moiety, and that there are disaccharides structurally related to the BLM disaccharide which are more efficient than the natural disaccharide at tumor cell targeting/uptake. Because BLM sugars can deliver molecular cargoes selectively to tumor cells, and thus potentially form the basis for a novel antitumor strategy, it seemed important to consider additional structural features capable of affecting the efficiency of tumor cell recognition and delivery. These included the effects of sugar polyvalency and net charge (at physiological pH) on tumor cell recognition, internalization, and trafficking. Since these parameters have been shown to affect cell surface recognition, internalization, and distribution in other contexts, this study has sought to define the effects of these structural features on tumor cell recognition by bleomycin and its disaccharide. We demonstrate that both can have a significant effect on tumor cell binding/internalization, and present data which suggests that the metal ions normally bound by bleomycin following clinical administration may significantly contribute to the efficiency of tumor cell uptake, in addition to their characterized function in DNA cleavage. A BLM disaccharide-Cy5** conjugate incorporating the positively charged dipeptide d-Lys-d-Lys was found to associate with both the mitochondria and the nuclear envelope of DU145 cells, suggesting possible cellular targets for BLM disaccharide–cytotoxin conjugates.
In a recent study, we demonstrated that structurally compact fluorophores incorporated into the side chains of amino acids could be introduced into dihydrofolate reductase from E. coli (ecDHFR) with minimal disruption of protein structure or function, even where the site of incorporation was within a folded region of the protein. The modified proteins could be employed for FRET measurements, providing sensitive monitors of changes in protein conformation. The very favorable results achieved in that study encouraged us to prepare additional fluorescent amino acids of potential utility for studying protein dynamics. Presently, we describe the synthesis and photophysical characterization of four positional isomers of biphenyl-phenylalanine, all of which were found to exhibit potentially useful fluorescent properties. All four phenylalanine derivatives were used to activate suppressor tRNA transcripts, and incorporated into multiple positions of ecDHFR. All phenylalanine derivatives were incorporated with good efficiency into position 16 of ecDHFR, and afforded modified proteins which consumed NADPH at rates up to about twice the rate measured for wild type. This phenomenon has been noted on a number of occasions previously and shown to be due to an increase in the off-rate of tetrahydrofolate from the enzyme, altering a step that is normally rate limiting. When introduced into sterically accessible position 49, the four phenylalanine derivatives afforded DHFRs having catalytic function comparable to wild type. The four phenylalanine derivatives were also introduced into position 115 of ecDHFR, which is known to be a folded region of the protein less tolerant of structural alteration. As anticipated, significant differences were noted in the catalytic efficiencies of the derived proteins. The ability of two of the sizeable biphenyl-phenylalanine derivatives to be accommodated at position 115 with minimal perturbation of DHFR function is attributed to rotational flexibility about the biphenyl bonds.
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