Anthraquinones Inhibitors

Anthraquinones are compounds able to interact with the nucleotide-binding sites of enzymes such as dehydrogenases, kinases, and ATPases, due to their structural similarities with adenine nucleotides such as ATP, adenosine diphosphate (ADP), and nicotinamide adenine dinucleotide (NAD). An-thraquinones and xanthones from natural sources have several potential therapeutic applications, for instance as antiviral, antimicrobial, or anti-cancer drugs (Ali et al. 2000). The extended planar structure of these compounds possibly makes them cytotoxic in that they are able to act as DNA intercalators. Even if that makes questionable the employment of this class of compounds in clinical practice, one might take advantage of them as chemical tools to elucidate the different cellular functions and regulations of CK2 for the optimization of highly specific and selective inhibitors.

Several anthraquinones and related xanthones have been tested with in vitro phosphorylation assays as potential inhibitors for CK2 (Sarno et al. 2002). Emodin (1,3,8-trihydroxy-6-methyl-antraquinone), previously reported as a tyrosine kinase inhibitor (Chan et al. 1993), was the first member of the anthraquinone family to be discovered that was also able to inhibit CK2 (Yim et al. 1999). The chemical formula and the inhibition potency (inhibitory constant, Ki) for emodin as well for other anthraquinones is reported in Fig. 5. Starting from this parent compound, a large number of anthraquinones and xanthone derivatives have been screened on CK2, allowing the identification of some interesting compounds with lower IC50 (or Ki) values. The most promsising ones have been submitted to crystallization trials, and for those that have generated good diffracting crystals (Fig. 5), the

Fig. 5 Chemical formulae and inhibition potencies for the four anthraquinones crystallized in complex with CK2. Emodin, 1,3,8-trihydroxy-6-methyl-antraquinone; MNA, 1,8-dihydroxy-4-nitro-anthraquinone; MNX, 1,8-dihydroxy-4-nitro-xanthen-9-one; DAA, 1,4-diamino-5,8-dihydroxy-anthraquinone

three-dimensional structures have been determined. The inhibitory constant K for the four compounds crystallized is in the micromolar range, from 1.85 mM for emodin to 0.35 mM for DAA.

Emodin is the first anthraquinone derivative crystallized in complex with Zea mays CK2 as an ATP site-directed competitive inhibitor. This natural compound is extracted from the rhizomes of Rheum palmatum and is used as anti-inflammatory and anti-cancer drug, especially in the Asian continent (Yim et al. 1999). Emodin has an inhibitory constant (Ki) of 1.85 mM for CK2, while it is poorly effective on other Ser/Thr protein kinases. This compound is able to inhibit also some Tyr-kinases, although with a lower efficiency (Jayasuriya et al. 1992). For instance, the receptor Tyr-kinase Her-2 neu is inhibited with a value of 21 ^M (Zhang et al. 1998), an order of magnitude higher than that for CK2.

Crystals of the emodin/CK2 complex were obtained by the soaking method (Battistutta et al. 2000). As emodin is poorly soluble in aqueous media, crystals of the maize CK2 catalytic subunit in the apo-form have been soaked for several hours with a saturated solution of emodin, which results in red for the presence of the inhibitor. The formation of the complex was evident because, within a few hours, the red coloring concentrated in the crystals, whereas the surrounding solution changed to pale orange. These crystals are isomorphous with those of maize CK2 grown in the presence of co-substrates ATP and GTP and also with those of the other anthraquinones complexes obtained with co-crystallization procedures (see below); all belongs to the C2 space group, with cell parameters reported in Table 1.

The co-substrate-competitive inhibitory activity of emodin is due to its ability to bind to the active site of CK2. Although the binding of the inhibitor poorly influences the overall architecture of the protein that remains in a structurally (but obviously not functional) active state, some regions of the catalytic subunit undergo appreciable structural changes, especially in the N-terminal lobe. The rms deviation for the 112 a-carbon atoms of the N-ter-

Table 1 Space group, unit cell parameters, and maximum resolution of diffraction for crystals of CK2 in the Apo form and in complex with inhibitors described in this chapter

CK2 complexes

Space group a, b, c (A)

a, b, g (°)

Resolution (A)

Apo-CK2

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