Interpretation of Protein-Drug Interaction through Fraction Bound and Relative Contribution of Secondary Sites

Mais, Roland F.; Keresztes‐Nagy, Steven; Zaroslinski, John F.; Oester, Y. T.

doi:10.1002/jps.2600630919

Cited by 37 publications

(5 citation statements)

References 14 publications

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“…As albumin binds MTX (Goldman, 1971;Liegler et al, 1969;Steele et al, 1979) biphasically and with relatively low affinities [71 X 104 M"' and 0.18 X 104 M_1 (Steele et al, 1979)], it probably contributes significantly to the high degree of nonspecific binding of radiolabeled folate analogues to L1210 cells (Henderson et al, 1980b). Salicylate also binds to albumin (Geddes & White, 1979;Mais et al, 1974), although there is some discrepancy in the reported association constants, which range from 8.6 X 103 M'1 (Mais et al, 1974) to about 5.6 X 105 M_1 (Geddes & White, 1979). Since the reactive moiety of the photoprobe is a salicylate derivative (/t-azidosalicylate), it is not unexpected that an excess of unlabeled MTX will not totally block labeling of albumin because labeling probably occurs at both the MTX and salicylate binding sites.…”

Section: Discussionmentioning

confidence: 99%

Photoaffinity analogs of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

Price

Freisheim²

1987

Biochemistry

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A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a Kt of 506 +/- 79 nM and a Vmax of 17.9 +/- 4.2 pmol min-1 (mg of total cellular protein)-1. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate (I50 = 1.0 microM). The parent compounds of the iodinated photoprobe inhibit [3H]methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a Ki of 66 +/- 21 nM. UV irradiation, at 4 degrees C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the Kt for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. In addition, no labeling occurs when a cell line that has a defective methotrexate transport system is similarly treated. Evidence that, in the absence of irradiation and at 37 degrees C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (Mr 38K and 21K) derived from the cell homogenate supernatant.

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Section: Discussionmentioning

confidence: 99%

Photoaffinity analogs of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

Price

Freisheim²

1987

Biochemistry

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“…The Scatchard plot (Fig. 5B) of specific STX binding was nonlinear and was well fit as the sum of 2 separate populations of binding sites (Mais et al, 1974).…”

Section: Functional Properties Of Voltage-sensitive Na Channels In Comentioning

confidence: 91%

Development of saxitoxin-sensitive and insensitive sodium channels in cultured neonatal rat astrocytes

Yarowsky

Krueger

1989

J. Neurosci.

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Voltage-sensitive Na channels were studied in cultures of neonatal rat cortical astrocytes. These channels were present at all times in culture as determined by tracer 22Na+ influx in the presence of batrachotoxin (BTX) and sea anemone polypeptide toxin (AxTx). The affinity of saxitoxin (STX) binding and sensitivity to STX inhibition of sodium influx were utilized to characterize these channels. Up to 7 d in culture, high-affinity 3H-STX binding (Kd of 0.2 nM at 4 degrees C) was very low, and 22Na+ influx was inhibited only by high concentrations (Ki = 170 nM) of STX. From 7 to 14 d, total specific binding of STX increased to a maximum of over 2 pmol/mg protein and remained constant for 28 d. By 14 d, inhibition of 22Na+ influx by STX was clearly biphasic, indicating the presence of 2 populations of channels with Ki's of 0.2 nM and 150 nM. At 14 d in culture, binding of 3H-STX to astrocyte membranes revealed the presence of 2 specific sites. During this second week, increasing numbers of high-affinity STX binding sites and increasing sensitivity to the inhibition of BTX + AxTx-stimulated 22Na+ influx by STX coincided with the change in morphology of primitive flat polygonal cells to highly branched stellate forms characteristic of mature astrocytes in vivo. Changes in culture conditions modified the time course of the onset of high STX affinity binding. Twenty-four hours after changing to serum-free G5 medium, there was both an 8-fold increase in STX binding sites and a change to a stellate shape in all cells. The results suggest that although low-affinity STX Na channels are always present in astrocytes, after 7 d in culture a different population of channels appears with the high affinity for STX characteristic of adult neuronal sodium channels. This spontaneous process is greatly accelerated by changing to a chemically defined medium.

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“…The Scatchard (1949) plot of R'/C against R', where C is concentration of free drug and R' is the molar ratio of DNA-bound drug (Cb) to DNA base-pair, showed non-linear curves, as exemplified in Figure 4. Thus the results were analysed using the following two-classes-of-sites model (Complex I and II), where two independent nonco-operative binding sites are postulated (Mais et al, 1974;Rosenthal, 1967):…”

Section: Absorption Binding Studiesmentioning

confidence: 99%

DNA-binding characterization of a novel anti-tumour benzo[a]phenazine derivative NC-182: spectroscopic and viscometric studies

Tarui

Doi

Ishida

et al. 1994

Biochemical Journal

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NC-182 is a novel anti-tumour compound having a benzo[a]phenazine ring. Fluorescence, absorption and c.d. spectroscopy, as well as viscometric titrations, were systematically performed to investigate the interaction mode of this drug with DNA and its effect on DNA conformation, based on comparative measurements with distamycin (DNA minor-groove binder) and daunomycin (DNA-base intercalator). NC-182 was found to be a potent intercalator of DNA, especially the B-form DNA, although no specificity was observed against the base-pair. The binding of NC-182 to B-DNA behaves biphasically, depending on the molar ratio (r) of drug to DNA: NC-182 acts to render the B-form structure rigid at relatively low r value and to promote the transformation of B- to non-B forms at high r values. It was also shown that NC-182 promotes the unwinding of Z-form DNA to B-form. Viscometric, u.v. 'melting' and c.d. experiments further showed that (1) the DNA duplex structure is thermally stabilized by intercalation with NC-182 and (2) the intercalation of NC-182 into a poly(dA).2poly(dT) DNA structure thermally stabilizes the triplex structure, resulting in a melting point close to that of the duplex structure; the melting curves of triplex and duplex structures coincide at r > 0.06. These observations make a significant contribution to our understanding of the biological properties of this novel benzo[a]phenazine derivative, a new anti-tumour tumour agent against multidrug-resistant and sensitive tumours.

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Interpretation of Protein-Drug Interaction through Fraction Bound and Relative Contribution of Secondary Sites

Cited by 37 publications

References 14 publications

Photoaffinity analogs of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

Photoaffinity analogs of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

Development of saxitoxin-sensitive and insensitive sodium channels in cultured neonatal rat astrocytes

DNA-binding characterization of a novel anti-tumour benzo[a]phenazine derivative NC-182: spectroscopic and viscometric studies

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