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Secondary Screens

2.3.1.1 GPC Spin Column/ESI-MS Drug Screening Demonstration Papers

A number of authors demonstrated the early use of the GPC spin column/ESI-MS methodology as a valid way to screen for compounds non-covalently bound to a target protein [11-13, 15]. The behavior of a target protein MMP-1, with a known binding hydroxyamide compound WY252, was evaluated as a singleton and in the presence of a mixture of non-binding compounds. In both cases, no significant difference was observed in the ability to detect the known binder in the mixture. A dramatic illustration of the ability of the GPC spin column/ESI-MS assay to analyze a mixture (in the negative ionization mode) has been demonstrated with MMP-1 protein and ten known hydroxyamide inhibitors with an IC50 range of 9 nM to 7.1 mM (Fig. 2.12A). Despite the wide range of IC50 values, all ten compounds were clearly observed in the ESI mass spectrum in the presence of MMP-1. None of the compounds were observed in the GPC spin column elu-ate when MMP-1 was not present (Fig. 2.12B).

2.3.1.2 Estrogen Receptor Target

A secondary screen for compounds that bind non-covalently to estrogen receptor (ER, MW 67 kDa) was evaluated and illustrated for 17b-estradiol (Kd @ 1 nM, MW 272 Da), the control compound in the study (Fig. 2.13), and WY234 (Kd 5 mM, MW 253 Da; Fig. 2.14). 17b-Estradiol is a relatively less polar material and was studied in the negative atmospheric pressure chemical ionization (APCI) mode since it produced a weak APCI spectrum in the positive mode and no spec-

Fig. 2.12 ESI (negative ionization mode) mass spectral analysis of the GPC spin column eluate of a mixture containing ten known MMP-1 inhibitors (A) with MMP-1 and (B) without MMP-1 (background). The [M-H]1- ions for the ten compounds are indicated by solid circles (•) on the spectra.

The same absolute intensity scale is used for both panels. The mixture is composed of the compounds listed at the right of the figure with their corresponding IC50 values. Reprinted from reference [15] with permission from the American Chemical Society.

Fig. 2.13 Negative ionization APCI mass spectra of the GPC spin column eluates of 20 |mM 17b-estradiol (MW 272 Da, Kd @ 1 nM) titrated with a variety of ER concentrations (1.25, 2.5, 5.0, 10.0, 20.0 |mM). A miniature P6 GPC spin column was used with 10-|mL samples, of which 2 |mL was injected into the APCI TOF mass spectrometer under low flow conditions (15 |L min-1 ). Note that the high resolution capability of the TOF instrument resolves the 17b-estradiol peak from the lower nominal-mass chemical noise. The same absolute intensity scale is used for all panels. The masses and intensities of the peaks are labeled.

Fig. 2.13 Negative ionization APCI mass spectra of the GPC spin column eluates of 20 |mM 17b-estradiol (MW 272 Da, Kd @ 1 nM) titrated with a variety of ER concentrations (1.25, 2.5, 5.0, 10.0, 20.0 |mM). A miniature P6 GPC spin column was used with 10-|mL samples, of which 2 |mL was injected into the APCI TOF mass spectrometer under low flow conditions (15 |L min-1 ). Note that the high resolution capability of the TOF instrument resolves the 17b-estradiol peak from the lower nominal-mass chemical noise. The same absolute intensity scale is used for all panels. The masses and intensities of the peaks are labeled.

trum in either the positive or negative ESI modes. WY234 is a relatively polar material that produced spectra in both the positive and negative ESI modes. Both compounds were titrated with ER. The stronger binding 17b-estradiol exhibited a strong response at a molar ratio of 5 mM ER/20 mM 17b-estradiol (on the resolved shoulder of a chemical background peak at the same nominal m/z of 271 [M-H]while the weaker binding WY234 exhibited a strong response at the higher molar ratio of 60 mM ER/300 mM WY234 in both the positive and negative ESI modes.

2.3.1.3 Non-covalent Binding of Drugs to RNA/DNA Targets

The GPC spin column/ESI-MS method has been applied to a number of RNA problems of pharmaceutical interest as an expedient and sensitive method in drug development strategies involving RNA-metabolizing enzymes [23]. These include: (i) the profiling of drug candidates to identify ones that do not bind to

Fig. 2.14 Positive and negative ionization ESI mass spectra of the GPC spin column eluates ofWY234 (MW 253 Da, Kd @ 1 ||M), a weak non-covalent binder, with ER, initially prepared at a variety of [WY234]/[ER] molar ratios. A miniature P6 GPC spin column was used with 10 |L samples. The same absolute intensity scale is used for all panels. The masses and intensities of the peaks are labeled.

Fig. 2.14 Positive and negative ionization ESI mass spectra of the GPC spin column eluates ofWY234 (MW 253 Da, Kd @ 1 ||M), a weak non-covalent binder, with ER, initially prepared at a variety of [WY234]/[ER] molar ratios. A miniature P6 GPC spin column was used with 10 |L samples. The same absolute intensity scale is used for all panels. The masses and intensities of the peaks are labeled.

RNA, (ii) the screening for antiviral compounds that do not bind to RNA but bind specifically to target RNA polymerases, (iii) the evaluation of the binding of aminoglycosides to RNA, and (iv) the evaluation of the binding of DNA intercalators and minor groove binders to RNA.

Certain classes of drugs may be detrimental as therapeutic agents if binding to RNA/DNA is an undesirable secondary side-effect. Such molecules are very likely to be cytotoxic to cells by interfering with the cellular machinery for DNA replication, DNA transcription and RNA translation. Often the inhibition of an enzyme in an in vivo cell-based assay is due to the interaction of the drug candidate with the cell's RNA/DNA resulting in false positive results. To eliminate such results and identify only those drug candidates which react with RNA/ DNA, a high throughput procedure was evaluated using the GPC spin column/ ESI-MS method for screening pharmaceutical candidates by studying their interaction with model duplex and single stranded RNAs. Drug candidates that bind non-covalently to RNA [23] or DNA can thereby be profiled either as single compounds or more efficiently as mixtures.

Three principal experiments were performed to identify potential drug candidates that bind non-covalently to RNA. The RNA/drug studies were performed under dilute and concentrated conditions using ethidium bromide, a known binder (intercalator) to RNA, as a reference RNA binding compound to validate the experimental strategy. In the first experiment, the formation of molecular ions of the compounds was ascertained under flow injection analysis in both the positive and/or negative ESI-MS ionization modes. In the second experiment, the GPC spin column eluates, recovered after incubation of the drug candidate in the buffer solution without RNA present, were analyzed by ESI-MS. Under these conditions the compounds should be fully retained in the GPC columns, which have a MW cutoff of about 6000 Da. However, any low-level detection of compounds provides a measure of ''noise'' for gauging false-positive controls. In the third experiment, the eluates recovered from reactions of the compounds with RNA were analyzed by ESI-MS. A biochemical control experiment showed that, under these conditions, the RNA is quantitatively recovered from the GPC spin column. However, the 125-mer RNA (MW 38 641 Da) does not produce an ESI mass spectrum in either the positive or negative ion modes. Compounds which passed through the GPC spin column due to non-covalent binding with the RNA, and which in the absence of RNA were retained by the column, would be flagged as unsuitable for further drug development. Using this technology, mixtures of drug candidates were analyzed, demonstrating a high throughput format for compound analysis. Figure 2.15 illustrates the positive ion ESI mass spectra obtained for a five-component mixture consisting of four drug candidates and ethidium bromide as a control under dilute conditions. Figure 2.15A illustrates the mass spectrum obtained under flow injection conditions, exhibiting molecular ions for ethidium bromide ([M]1+: m/z 314) and drug candidate WY311 ([M]1+: m/z 319). The other three components ionize in the negative ion mode but not in the positive ion mode. Figure 2.15B illustrates the mass spectrum obtained for the GPC spin column eluate of the mixture (in the absence of RNA). No ions were observed, indicating that the GPC spin column retained all the compounds. Figure 2.15C illustrates the mass spectrum obtained for the GPC spin column eluate of the mixture incubated in the presence of RNA. As expected ethidium bromide, which non-covalently binds to RNA, passed through the GPC spin column. However WY311, which was incubated with RNA, did not pass through the GPC spin column. The three negative ion compounds also do not pass through the GPC spin column in the absence and presence of RNA. Therefore, all four drug candidates have desirable pharmaceutical profiles in that they do not form non-covalent RNA:drug complexes.

RNA/drug studies with antiviral agents that target-specific RNA polymerases were conducted under dilute and concentrated conditions, viz. 0.25 mM RNA/30 mM drug and 10 mM RNA/300 mM drug, respectively, where the model RNA was a 125-mer with a MW of 38 641 Da. Ethidium bromide, a known binder (intercala-tor) to RNA, was used to validate the experiments under dilute and concentrated

Fig. 2.15 Positive ion ESI mass spectra under flow injection conditions for a five-component mixture. (A) Mass spectrum for the direct analysis of the mixture of which only two components ionize in the positive ion mode: ethidium bromide (MW 314 Da) and WY311 (MW 319 Da). (B) Mass spectrum of the GPC spin column eluate for the mixture without RNA present. Neither ethidum bromide or WY311 passed through the GPC spin column. (C) Mass spectrum of the GPC spin column eluate of the mixture incubated with RNA. In the presence of RNA, ethidium bromide passed through the GPC spin column as a non-covalent complex with RNA while WY311 did not pass through the GPC spin column and did not form a non-covalent complex with RNA.

Fig. 2.15 Positive ion ESI mass spectra under flow injection conditions for a five-component mixture. (A) Mass spectrum for the direct analysis of the mixture of which only two components ionize in the positive ion mode: ethidium bromide (MW 314 Da) and WY311 (MW 319 Da). (B) Mass spectrum of the GPC spin column eluate for the mixture without RNA present. Neither ethidum bromide or WY311 passed through the GPC spin column. (C) Mass spectrum of the GPC spin column eluate of the mixture incubated with RNA. In the presence of RNA, ethidium bromide passed through the GPC spin column as a non-covalent complex with RNA while WY311 did not pass through the GPC spin column and did not form a non-covalent complex with RNA.

conditions. In studies of seven antiviral drug candidates, three compounds at high concentrations exhibited binding to the 125-mer RNA while four compounds exhibited no binding under dilute and concentrated conditions. These results demonstrate that the latter four compounds, which under the different concentration conditions did not bind to the 125-mer RNA, are preferable anti-viral drug candidates.

Similar RNA-binding studies were performed with a variety of aminoglycosides and DNA-binding compounds, using ethidium bromide as a control. Table 2.3 summarizes the GPC spin column/ESI-MS results for paromomycin (an aminoglycoside) and DAPI (a DNA-binding compound), which both bind weakly to the 125-mer RNA. Distamycin (a DNA-binding compound) did not bind to the RNA, whereas ethidium bromide did. The results obtained in these studies further demonstrate that the detection of GPC spin column eluates with ESI-MS can be used successfully to screen, in a high throughput fashion, drug candidates that non-covalently bind to RNA. Likewise, these same procedures can be used to screen compounds that non-covalently bind to DNA.

Table 2.3 GPC spin column/ESI-MS non-covalent binding studies of RNA with model RNA and DNA binding compounds (intercalators).

(I) No GPC spin column; (II), (III) Through GPC spin column

(I) No GPC spin column; (II), (III) Through GPC spin column

Table 2.3 GPC spin column/ESI-MS non-covalent binding studies of RNA with model RNA and DNA binding compounds (intercalators).

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