Research ArticleCHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Biospecific Interaction Analysis (BIA) of Low-Molecular Weight DNA-Binding Drugs

Roberto Gambari, Giordana Feriotto, Cristina Rutigliano, Nicoletta Bianchi and Carlo Mischiati
Journal of Pharmacology and Experimental Therapeutics July 2000, 294 (1) 370-377;

Abstract

DNA-binding drugs have been reported to be able to interfere with the activity of transcription factors in a sequence-dependent manner, leading to alteration of transcription. This and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases and viral infections. The analysis of the biological activity of DNA-binding drugs by footprinting, gel retardation, polymerase chain reaction, and in vitro transcription studies does not allow a real time study of binding to DNA and dissociation of the generated drugs/DNA complexes. The recent development of biosensor technologies for biospecific interaction analysis (BIA) enables monitoring of a variety of molecular reactions in real-time by surface plasmon resonance (SPR). In this study, we demonstrate that molecular interactions between DNA-binding drugs (chromomycin, mithramycin, distamycin, and MEN 10567) and biotinylated target DNA probes immobilized on sensor chips is detectable by SPR technology using a commercially available biosensor. The target DNA sequences were synthetic oligonucleotides mimicking the Sp1, NF-kB, and TFIID binding sites of the long terminal repeat of the human immunodeficiency type 1 virus. The results obtained demonstrate that mithramycin/DNA complexes are less stable than chromomycin/DNA complexes; distamycin binds to both NF-kB and TATA box oligonucleotides, but distamycin/(NF-kB)DNA complexes are not stable; the distamycin analog MEN 10567 binds to the NF-kB mer and the generated drug/DNA complexes are stable. The experimental approach described in this study allows fast analysis of molecular interactions between DNA-binding drugs and selected target DNA sequences. Therefore, this method could be used to identify new drugs exhibiting differential binding activities to selected regions of viral and eukaryotic gene promoters.

Recent published observations from a large number of molecular biology laboratories demonstrate that DNA-binding drugs, including distamycin (Broggini et al., 1989; Gambari et al., 1991; Dorn et al., 1992;Feriotto et al., 1994), chromomycin (Bianchi et al., 1996), mithramycin (Snyder et al., 1991), actinomycin D (Vaquero and Portugal, 1998), elsamicin A (Vaquero and Portugal, 1998), and CC-1065 (Welch et al., 1994), interfere in a sequence-dependent manner with biological functions such as restriction enzyme cleavage of DNA, topoisomerase activity, protein-DNA interactions, in vitro and in vivo transcription (Van Dyke and Dervan, 1983; Broggini et al., 1989; Gambari et al., 1991; Dorn et al., 1992; Feriotto et al., 1994, Gambari and Nastruzzi, 1994). For instance, the G+C selective DNA binding drugs mithramycin and chromomycin (Van Dyke and Dervan, 1983) suppress molecular interactions between transcription factor Sp1 and viral and eukaryotic promoters, leading to an alteration of the expression of Sp1-regulated transcriptional units, including the oncogene Ha-ras, the collagen-α1(I) gene and HIV-1 (Snyder et al., 1991; Bianchi et al., 1996; Vaquero and Portugal, 1998). By contrast, the A+T selective DNA binding drug distamycin does not interfere with the interaction between Sp1 and target DNA sequences, being able on the contrary to fully suppress binding of TF-IID/TBP to the TATA-box element of the HIV-1 long terminal repeat (LTR) (Feriotto et al., 1994). These and similar effects could have important practical applications in the experimental therapy of many human pathologies, including neoplastic diseases (Vaquero and Portugal, 1998) and AIDS (Gambari and Nastruzzi, 1994). Following this hypothesis, a large number of investigations on the design, synthesis, and characterization of the potential sequence-selectivity of DNA binding drugs was recently published using DNase I footprinting, gel retardation studies, polymerase chain reaction, and in vitro transcription (Portugal and Waring, 1987;Broggini et al., 1989; White and Phillips, 1989; Churchill et al., 1990; Fox et al., 1990; Laughton et al., 1990; Gambari et al., 1991;Montecucco et al., 1991; Snyder et al., 1991; Dorn et al., 1992; Chiang et al., 1994; Feriotto et al., 1994; Passadore et al., 1994, 1995;Welch et al., 1994; Bellorini et al., 1995; Bianchi et al., 1996;Vaquero et al., 1998). It should be stressed that most of these techniques are steady-state methodologies and therefore are not suitable for an easy determination of 1) the binding efficiency of DNA-binding drugs to target DNA elements and 2) the stability of drugs-DNA complexes. Of course, these parameters are of great interest, because they could be intimately associated with the biological activity of DNA-binding drugs.

The recent development of biosensor technologies for biospecific interaction analysis (BIA) (Jonsson et al., 1991; Vadgama and Crump, 1992; Malmqvist, 1993) enables monitoring of a variety of molecular reactions in real-time by surface plasmon resonance (SPR). This optical technique detects and quantifies changes in refractive index in the vicinity of the surface of sensor chips to which ligands are immobilized (Vadgama and Crump, 1992; Malmqvist, 1993). Because the changes in the refractive index are proportional to the changes in the adsorbed mass (Malmqvist, 1993), the SPR technology allows detection of biomolecules (analytes) interacting with the ligand immobilized on the sensor chip. For instance, if the ligand is a biotinylated single-stranded DNA, SPR technology could monitor DNA-DNA hybridization in the same time as it occurs (Wood, 1993; Nilsson et al., 1995). SPR technology was also applied to studies on protein-protein interactions, protein-DNA recognition, antibody-antigen binding, as well as to a large variety of biomolecular applications involving high-molecular weight molecules (Malmqvist, 1993). In sharp contrast, few reports were published on BIA analysis on low-molecular weight compounds, and to our knowledge, no data are available in the literature on the possible use of SPR technology to detect differential interactions between DNA-binding drugs and target DNA sequences. It is well known that detection of molecular interactions of low-molecular weight analytes (<2000 Da) is expected to be difficult, especially if the analyte is not pure. This is largely due to the low expected increase of resonance units (RU) following binding of a low-molecular weight (200–1000 Da) analyte to its immobilized ligand (Karlsson and Stahlberg, 1995). For these reasons, the demonstration of the possible use of BIA analysis for studies on DNA-binding drugs could be of interest.

In this paper we used the biosensor BIAcore (Pharmacia Biosensors, Uppsala, Sweden) to detect, by BIA and SPR technology, the binding activity of two G+C selective minor groove ligands, mithramycin and chromomycin, and two A+T selective binders, distamycin and MEN 10567. As a model system, we selected three oligonucleotides mimicking the binding sites of transcription factors Sp1, NF-kB and TFII-D, present within the LTR of HIV-1. These oligonucleotides were immobilized on streptavidin-coated sensor chips and used as ligands. The results obtained show that SPR technology is a suitable one-step, nonradioactive approach to demonstrating DNA-binding activity of low molecular minor groove ligands and stability of drugs-DNA complexes.

Materials and Methods

DNA-Binding Drugs and Synthetic Oligonucleotides.

Chromomycin (1183.3 Da), mithramycin (1085.2 Da), and distamycin A (518 Da) were purchased from Sigma Chemical Co. (St. Louis, MO). MEN 10567 (639 Da) was obtained from Menarini Research (Florence, Italy). Chemical structures of these compounds are shown in Fig. 1. The Sp1 (7507 Da), NFkB (8537 Da) and TATA (5503 Da) synthetic oligonucleotides are shown in Table 1 and were purchased from Pharmacia (Uppsala, Sweden). All stock solutions of DNA-binding drugs (1.2 mM) were stored at −20°C in the dark and diluted immediately before use in CH/MT buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 20 mM MgCl2).

Figure 1
Figure 1

Chemical structures of mithramycin, chromomycin, distamycin and MEN10567.

View this table:
Table 1

Synthetic oligonucleotides used in this study

BIA Using Biosensor Technology.

The BIAcore-1000 instrument (Pharmacia Biosensors) was used in all experiments (Jonsson et al., 1991; Vadgama and Crump, 1992; Malmqvist, 1993). Sensor chip SA5 (research grade), precoated with streptavidin was purchased from Pharmacia Biosensors. The experiments were conducted at 25°C temperature and at 4 μl/min flow rate (Nilsson et al., 1995). Rapid capture of about 700 to 900 RU of biotinylated Sp1, NF-kB, and TATA single-stranded DNA (0.5 μg/50 μl of HBS buffer, 30-μl injection) (HBS buffer = 10 mM Hepes, pH 7.4, 0.15 M NaCl, 3.4 mM EDTA, and 0.05% Surfactant P2; Pharmacia Biosensors) was reproducibly obtained within 4 to 6 min (data not shown). Double-stranded Sp1, NF-kB, and TATA mers were generated by a 20- to 30-μl injection of the complementary oligonucleotides (Sp1c, NF-kBc, and TATAc mers, 0.5 μg/50 μl of HBS). The obtained double-stranded DNAs were found to be stably immobilized on SA5 sensor chips, as 30-μl injections of running buffer did not cause a decrease of the SPR signals (data not shown). The binding of minor groove ligands to double-stranded target DNA sequences was monitored after a 20- to 30-μl injection of 0.125 to 5 μM compounds. Running buffers were HBS buffer and CH/MT buffer (100 mM NaCl, 50 mM Tris, pH 7.4, 20 mM MgCl2). Buffers were filtered and degassed before use. Generation of double-stranded target DNA sequences was performed in HBS buffer. Analysis of the obtained sensorgrams was performed using the BIA-evaluation Software. Suitable blank control injections with running and/or injection buffers were performed, and the resulting sensorgrams subtracted from the experimental sensorgrams. The apparent stoichiometry of the surface complex (i.e. the number of analyte molecules that can bind to one ligand molecule) was calculated from the saturating binding capacity of the surface by the equation (Jonsson et al., 1991; Vadgama and Cump, 1992; Malmqvist, 1993):stoichiometry=analyte responseligand response×ligand mol.wt.analyte mol.wt.

Results

Detection of Binding of Mithramycin to the SA-Sp1 Sensor Chip.

To determine whether binding of mithramycin to DNA is detectable by SPR technology, the experiments shown in Fig.2 were performed. We first produced a SA5 sensor chip containing Sp1 target double-stranded DNA. Second, we allowed mithramycin binding to (Sp1)DNA. Third, we studied stability of mithramycin-(Sp1)DNA complexes by injecting binding buffer.

Figure 2
Figure 2

A, binding of mithramycin to double-stranded HIV-1 Sp1 DNA. This experiment was conducted with CH/MT buffer as running buffer. Sp1c mer was injected in HBS buffer (A,a). The sensorgram shows that injection of 20 μl of HIV-1 LTR Sp1b mer (0.25 μg/50 μl of CH buffer) generate an increase (A,a) in RU. The binding of mithramycin to the SA5 sensor chip containing double-stranded HIV-1 LTR Sp1 DNA is shown in A,b. An increase of 833 RU was observed following a 30-μl injection of 5 μM mithramycin. The decrease of RU during the washing step (injection of CH/MT binding buffer) is shown in A,c. B, after injection of Sp1c mer, a 20-μl injection of increasing concentrations (a = 0.5 μM; b = 1 μM; c = 2 μM; d = 4 μM; e = 5 μM; f = 8 μM) of mithramycin was performed. The sensorgrams have been elaborated using the BIA-evaluation Software (Pharmacia Biosensors). C, relationship between bound mithramycin (RUmax − RUi) and amount of ligand Sp1 mer (RUi − RUo) present on the flow cell surface. In this experiment, injections of 20 μl of 5 μM mithramycin were performed to different flow cells containing increasing amounts of target Sp1 mer.

Double-stranded Sp1 target DNA was usually generated by 20-μl injections of 500 ng of complementary Sp1 oligonucleotide (Sp1c mer) (see Table 1 for nucleotide sequences) in 50 μl of HBS buffer. When binding of mithramycin was conducted in a CH/MT buffer lacking MgCl2 (100 mM NaCl, 50 mM Tris, pH 7.4), no increase of RU was detected (data not shown), in agreement with results reported elsewhere (Dervan, 1986; Sastry et al., 1995), showing that binding of these ligands to target DNA requires Mg2+. Therefore, in this and in all the experiments performed with mithramycin and chromomycin, CH/MT buffer, containing 20 mM MgCl2, was used as binding and washing buffer. In these experimental conditions, preliminary experiments demonstrated that mithramycin does not bind to single-stranded Sp1 mer (data not shown). By sharp contrast, Fig. 2A demonstrates that, after double-stranded Sp1-mer formation (Fig. 2A, a), a 30-μl injection of 5 μM mithramycin leads to a sharp increase of RU (Fig. 2A, b). The value of RUmax − RUi was indeed found to be in this experiment 833 RU. These data demonstrate that mithramycin binding to immobilized double-stranded Sp1 DNA is detectable by SPR and biosensor technologies. However, from the sensorgram shown in Fig. 2A, it appears clear that the mithramycin/DNA complexes are not stable, because injection of binding buffer (Fig. 2A, c) leads to a sharp reduction of RU, reaching a level similar to that found before mithramycin injection (RUres − RUi = 71; 8.52% of RUmax − RUi).

In Fig. 2B, the binding of mithramycin to target Sp1 DNA was studied at different drug concentrations (0.5–8 μM). The experiment shows that the increase of RU is dose-dependent and reaches saturation at 4 to 6 μM mithramycin. The sensorgrams obtained were analysed using the BIA-simulation and the BIA-evaluation softwares. The values of RUmax − RUi for mithramycin were found to be, in nine different experiments, 825 ± 133 RU/1000 RU of Sp1c mer immobilized on the SA5 sensor chip. When the same concentration of mithramycin was injected to flow cells containing increased amounts of target Sp1 double-stranded oligonucleotide, a direct relationship was found between RUmax − RUi values and amounts of Sp1 mer immobilized on the sensor chip (RUi − RUo; Fig. 2C). This experiment is important when low-molecular weight analytes are used (Karlsson and Stahlberg, 1995) and indicate that the observed increases of RUmax (Fig. 2, A and B) are due to molecular interactions of mithramycin to the Sp1 mer, rather than mass transport effects. The apparent stoichiometry of the surface complexes was calculated from the saturating binding capacity of the SA-Sp1 mer surface, considering 1085 and 7505 Da the molecular weights of the analyte (mithramycin) and ligand (Sp1c mer). The obtained data give evidence for a 6:1 (analyte:ligand) stoichiometry in most of the experiments performed (average 5.7:1). This finding is consistent with a large number of NMR studies indicating that mithramycin and the analogue chromomycin bind as a symmetric dimer to self complementary target hexanucleotide and octanucleotide complexes (Gao et al., 1992;Chen et al., 1997).

Detection of Binding of Distamycin to the SA-NFkB Sensor Chip.

To determine whether binding of distamycin to DNA is detectable by SPR technology, the experiment shown in Fig. 2 was repeated using a SA sensor chip carrying a double-stranded oligonucleotide containing the HIV-1 LTR NF-kB sequence. We previously demonstrated by DNase I footprinting experiments that distamycin binds to the NF-kB binding sites of the HIV-1 LTR (Feriotto et al., 1994b). The SA5 sensor chip containing NF-kB target double-stranded DNA was prepared as described for Sp1. Distamycin was injected in HBS buffer. The stability of distamycin/DNA complexes was studied with a further injection of HBS buffer.

Fig. 3A demonstrates that, after double-stranded NF-kB-mer formation (Fig. 3A, a), a 30-μl injection of 5 μM distamycin leads to a sharp increase of RU (Fig. 3A, b; RUmax − RUi = 147). However, the distamycin/(NF-kB)DNA complex is not stable, because injection of binding buffer causes a fast reduction in RU (RUres − RUi = 6). The binding kinetics of distamycin were studied at different drug concentrations; the results obtained are shown in Fig. 3B and indicate that the increase of RU is dose-dependent. The values of RUmax − RUi for distamycin were found to be, in eight different experiments, 300.4 ± 71 RU/1000 RU of NF-kBc mer immobilized on the SA5 sensor chip. Also, in the case of distamycin, control experiments were performed demonstrating a direct relationship between RUmax − RUi values and amounts of target NF-kB mer immobilized on the sensor chip (RUi − RUo; Fig. 3C). The apparent stoichiometry of the surface complex was calculated considering 518 and 8537 Da the molecular weights of the analyte (distamycin) and ligand (NF-kBc mer). The obtained data give evidence for a 5:1 (analyte:ligand) stoichiometry in most of the experiments performed (average 4.9:1), consistent with previously published observations showing that distamycin interacts with the minor groove of target DNA sequences in a dimeric state (Chen et al., 1997).

Figure 3
Figure 3

A, binding of distamycin to double-stranded HIV-1 NF-kB DNA. This experiment was conducted with HBS buffer as running buffer. NF-kBc mer was injected in HBS buffer (A,a). The sensorgram shows that injection of 20 μl of HIV-1 LTR NF-kBc mer (0.25 μg/50 μl of HBS buffer) generate an increase (A,a) in RU. The binding of distamycin to the SA5 sensor chip containing double-stranded HIV-1 LTR NF-kB DNA is shown in A,b. An increase of 147 RU was observed following a 30-μl injection of 5 μM distamycin. The decrease of RU during the washing step (injection of HBS binding buffer) is shown in A,c. B and C, after injection of NF-kBc mer, a 20-μl injection of increasing concentrations (a = 0.125 μM; b = 0.25 μM; c = 0.5 μM; d = 1 μM; e = 2 μM; f = 5 μM;) of distamycin was performed. The sensorgrams have been elaborated using the BIA-evaluation Software (Pharmacia Biosensors). C, relationship between bound distamycin (RUmax − RUi) and amount of ligand NF-kB mer (RUi− RUo) present on the flow cell surface. In this experiment, injections of 20 μl of 5 μM distamycin were performed to different flow cells containing increasing amounts of target NF-kB mer.

Differential Binding of Distamycin and Mithramycin to SA5-Sp1 and SA5-NF-kB Sensor Chips.

In the experiment shown in Fig. 4 (A and B), 5 μM mithramycin was injected onto SA5-Sp1 (Fig. 4A) and SA5-NF-kB (Fig. 4B) sensor chips. After mithramycin injection (A,a and B,a), we performed an injection of binding buffer (A,b and B,b). The results obtained demonstrate that binding of mithramycin to the SA5-Sp1 sensor chip is more efficient, although binding occurs also to the NF-kB sensor chip. In both cases, the drug/DNA complexes rapidly dissociate. The sequence-selective binding of distamycin is shown in Fig. 4 (C and D). In this experiment 5 μM distamycin was injected onto SA5-NF-kB (C) and SA5-Sp1 (D) sensor chips. After distamycin injection (C,a and D,a), we injected HBS binding buffer (C,b and D,b). The results obtained demonstrate that binding of distamycin to the SA5-Sp1 sensor chip does not occur, whereas binding to the NF-kB sensor chip is readily detectable.

Figure 4
Figure 4

Differential binding of mithramycin and distamycin to SA5-Sp1 and SA5-NF-kB sensor chips. 5 μM mithramycin (A, B) and 5 μM distamycin (C, D) were injected onto SA5-Sp1 (A, D) and SA5-NF-kB (B, C) sensor chips. After injection of DNA-binding drugs (Aa, Ba, Ca, and Da), a 15 μl injection of binding buffer (Ab, Bb, Cb, and Db) was performed. Binding buffers were CH/MT and HBS buffers in the case of mithramycin and distamycin, respectively.

Differential Binding of Distamycin to SA5-NF-kB and SA5-TATA Box Sensor Chips.

In the experiment shown in Fig. 5, 5 μM distamycin was injected onto SA5-NF-kB (Fig. 5A) and SA5-TATA box (Fig. 5B,b) sensor chips. After distamycin injection (A,b and B,b), we injected HBS running buffer (A,c and B,c). The results obtained demonstrate that binding of distamycin to the SA5-TATA box sensor chip is more efficient than binding to the NF-kB sensor chip. In addition, the data obtained clearly show that the distamycin/TATA box complexes are more stable than distamycin-NF-kB mer complexes. The values of RUres − RUi were found to be 4 (2.45% of RUmax − RUi) in the case of binding of distamycin to SA5-NF-kB flow cell; the values of RUres − RUi were found to be 117 (59.6% of RUmax − RUi) in the case of binding of distamycin to the TATA box flow cell. These results suggest that SPR technology and BIA are useful for the study of the activities of DNA-binding drugs toward different promoter sequences.

Figure 5
Figure 5

Differential binding of distamycin to SA5-NF-kB and SA5-TATA box sensor chips. Distamycin (5 μM) was injected onto SA5-NF-kB (A) and SA5-TATA box (B) sensor chips. After distamycin injection (Ab and Bb), a 15-μl injection of HBS running buffer (Ac and Bc) was performed. The sensorgrams have been elaborated using the BIA-evaluation Software (Pharmacia Biosensors).

Differential Stability of Drug/DNA Complexes Generated After Injection of Mithramycin and Chromomycin onto SA5-Sp1 Sensor Chip.

In the experiment shown in Fig. 6, 5 μM mithramycin (Fig. 6A,b) and chromomycin (Fig. 6B,b) were injected onto SA5-Sp1 sensor chips. After injection of DNA-binding drugs, we performed a 30-μl injection of CH/MT running buffer (Fig.6 A,c and B,c). The results obtained demonstrate that chromomycin/Sp1 complexes are more stable than mithramycin/Sp1 mer complexes. The values of RUres − RUi were 33 (6.18% RUmax − RUi) for mithramycin/Sp1 mer interactions and 531 (73.6% RUmax − RUi) for chromomycin/Sp1 mer interactions.

Figure 6
Figure 6

Differential stability of drug/DNA complexes generated after injection of mithramycin and chromomycin onto SA5-Sp1 sensor chip. Mithramycin (5 μM) (A) and chromomycin (5 μM) (B) were injected onto SA5-Sp1 sensor chips. After injection of DNA-binding drugs (Ab and Bb), a 30-μl injection of CH/MT running buffer (Ac and Bc) was performed. The sensorgrams have been elaborated using the BIA-evaluation Software (Pharmacia Biosensors).

Differential Binding of Distamycin Analogues to SA5-NF-kB Sensor Chip.

In the experiment shown in Fig. 7, 2 μM distamycin (Fig. 7A,b) and MEN 10567 (Fig. 7B,b) were injected onto the SA5-NF-kB sensor chip. In this experiment the washing step was performed with HBS running buffer (Fig. 7, A,c and B,c). The results obtained demonstrate that the dissociation of distamycin from NF-kB mer is fast, whereas MEN 10567 binds to the SA5-NF-kB sensor chip generating stable complexes. The values RUres − RUi were 10 (6.5% RUmax − RUi) for distamycin/NF-kB mer interactions and 158 (70.2% RUmax − RUi) for MEN 10567/NF-kB mer interactions.

Figure 7
Figure 7

Differential binding of distamycin analogues to SA5-NF-kB sensor chip. Distamycin (2 μM) (A) and MEN 10567 (2 μM) (B) were injected onto the SA5-NF-kB sensor chip. Binding and washing steps were performed with HBS running buffer. After injection of DNA-binding drugs (Ab and Bb), a 30-μl injection of HBS buffer (Ac and Bc) was performed. The sensorgrams have been elaborated using the BIA-evaluation Software (Pharmacia Biosensors).

Discussion

DNA-binding drugs have recently been the object of many investigations aimed at developing sequence-selective inhibitors of transcription (Chen et al., 1997). This issue is of great interest in order to design antitumor compounds and antiviral agents, as well as compounds able to induce cellular differentiation (Gambari and Nastruzzi, 1994; Nilsson et al., 1995). For instance, chromomycin binds to GC-rich DNA sequences (van Dyke and Dervan, 1983), inhibits the interaction between the transcription factor Sp1 and target DNA (Snyder et al., 1991; Bianchi et al., 1996), and blocks the transcription directed by promoters that are regulated by Sp1, such as SV40 (Ray et al., 1989), c-myc (Snyder et al., 1991), and HIV-1 (Bianchi et al., 1996). On the other hand, distamycin was found to bind A+T-rich gene sequences and to inhibit the interactions between Oct-1, GATA-1, TBP/TFIID, and Eryf-1 DNA-binding proteins to the relative DNA target sites (Broggini et al., 1989; Feriotto et al., 1994a; Welch et al., 1994; Bianchi et al., 1996).

The major conclusion gathered from the experiments reported in this paper is that interactions of DNA-binding drugs to target DNA sequences can be monitored in real-time using SPR technology and a commercially available biosensor. This is supported by the finding that the increase of RUmax − RUi values is proportional to the amounts of specifically recognized target DNA immobilized on the sensor chip after injection of a same concentration of DNA-binding drugs. This experimental approach has been shown to be useful for detection of low-molecular weight analytes as well as for characterization of weak interactions by BIA (Karlsson and Stahlberg, 1995). Please note that the data obtained during the association phases of the BIA experiments described in the present study are in agreement with previously published results. In particular, distamycin was shown to be able to interact with the NF-kB binding sites and the TATA-box element of the HIV-1 LTR, but unable to bind to the G+C-rich Sp1 binding sites. Furthermore, as found in the present study, mithramycin and chromomycin do not bind to single-stranded DNA and do require Mg2+ for efficient interaction with target Sp1 binding sites.

To our knowledge, this is the first report concerning the use of SPR and biosensor technology to compare real-time biospecific interactions between different DNA-binding drugs and specifically recognized target DNA elements. From the practical point of view, this experimental approach appears to be of great interest for the following reasons: (a) results are obtained within 1 h; (b) unlike footprinting and gel retardation, this technology does not have need of32P-labeled probes; and (c) BIA allows kinetics studies of both association and dissociation.

We think that this approach could be applied to develop drugs exhibiting differential affinity to target DNA sequences and differential ability to generate stable drugs/DNA complexes.

The results shown in the present study demonstrate indeed that BIA allows identification of different stabilities of molecular complexes generated by the interactions between target DNA sequences and different DNA-binding drugs exhibiting similar chemical structure [i.e., chromomycin and mithramycin (Fig.6); distamycin and MEN 10567 (Fig.7)]. In particular, it is interesting to point out that the different number of pyrroles (three in the case of distamycin, four in the case of MEN 10567) lead to a differential ability to generate stable complexes with the NF-kB binding sites of the HIV-1 LTR. In addition, BIA analysis demonstrates that a same DNA-binding drug (distamycin in our study) interacts with different target DNA sequences (the NF-kB and TATA box binding sites of the HIV-1 LTR) generating complexes that exhibit different stabilities (Fig. 5).

In conclusion, this report is one of the few reports demonstrating the possible use of biosensor technology and SPR to investigate molecular interactions involving low-molecular weight ligands (DNA-binding drugs) and target molecules (DNA). The determination of the stability of molecular complexes generated from the interactions between DNA-binding drugs and target DNA sequences is important in the design of antitumor and antiviral drugs exhibiting a well defined mechanism of action.

Footnotes

  • Send reprint requests to: Prof. Roberto Gambari, Department of Biochemistry and Molecular Biology Via L. Borsari n.46, 44100 Ferrara, Italy. E-mail: gam{at}dns.unife.it

  • ↵1 This work was supported by CNR PF Biotecnologie, by ISS(AIDS 1998), by PRIN-98, and by Ricerca Finalizzata 1999, Ministero della Sanità, Italy. The BIAcore-1000 was obtained with a grant from the “Grandi attrezzature ad uso comune” fund of Ferrara University. N.B. and C.M. are recipients of Fondazione Italiana Ricerca sul Cancro and Associazione Italiana Ricerca sul Cancro fellowships, respectively.

  • Abbreviations:
    LTR
    long terminal repeat
    BIA
    biospecific interaction analysis
    SPR
    surface plasmon resonance
    RU
    resonance unit(s)
    • Received June 1, 1999.
    • Accepted February 4, 2000.

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Research ArticleCHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Biospecific Interaction Analysis (BIA) of Low-Molecular Weight DNA-Binding Drugs

Roberto Gambari, Giordana Feriotto, Cristina Rutigliano, Nicoletta Bianchi and Carlo Mischiati
Journal of Pharmacology and Experimental Therapeutics July 1, 2000, 294 (1) 370-377;
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