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Phosphoinositide-3-kinases (PI3Ks) are a class of enzymes phospho-rylating lipids. In 1988, Cantley¹ found that PI3Ks phosphorylate the 3-hydroxy group in the myoinositol ring of phosphatidylinositol and produce phosphatidylinositol-3-phosphate. These lipids are members of eukaryotic cell membranes, where they play important roles in lipid and protein trafficking. The PI3Ks are divided into classes (I, II and III). The best known family member is the catalytic domain called p110α, which belongs to class Ia²(Fig. 2).

Natural Products from Terrestrial Microbial Organisms with Cytotoxic Cell Cycle Inhibitors 149

Figure 2 Classes of cell cycle inhibitors.

Class I: p110α, p110γ; Class II: PI3K-C2α; Class III: hVps34; Class IV: mTOR, ATM, ATR, DNA-PK

HEAT: a protein-protein interaction structure of two tandem anti-parallel a-helices found in Huntingtin, elongation factor 3 (EF3), the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor

FAT: a domain structure shared by FRAP, ATM and TRRAP FRB: FKBP12/rapamycin binding domain

FATC: FAT C-terminus

PI3Ks are negatively-regulated by PTEN (phosphatase and tensin homo-logue), which is deleted, mutated or repressed in cancer cells. Hence, the persistent activation of PI3K downstream pathways stimulates prolifera-tion by phosphorylaprolifera-tion of Akt (protein kinase B). Akt down-regulates the expression of death genes and activates NF-κB (nuclear factor kappa B) and other proteins important for cell survival. Akt2 is overexpressed in ovarian, breast and pancreatic cancers and Akt3 is increasingly found in breast and prostate tumors.

A PI3K-related family, also called PI3K superfamily or sometimes class IV,³ are the serine/threonine protein kinases, which consist of a similar

sequence to PI3Ks (Fig. 2).⁴ Members of this superfamily include ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia related), DNA-dependent protein kinases and mTOR (mammalian target of rapamycin).

Protein phosphorylation is an important mechanism in regulating the cell cycle. In the case of DNA damage, ATM and ATR activate p53 either directly or by phosphorylation of checkpoint kinases (Chk). Various PI3K inhibitors can be used to substitute for PTEN as a regulator and inhibit Akt activation and Chk phosphorylation. PI3K inhibitors interact with PI3K-related proteins as well (Table 1).

The first inhibitor derived from microbial origin was wortmannin (1) (Fig. 3).⁵ It was isolated from Penicillum funiculosum and belongs to the class of viridins, which consist of a steroid-like skeleton with a lactone in the A ring and a furan connected to the A and B ring.

Table 1 Inhibitor-reactive Lysine in Different P13Ks Protein Binding Amino Acids Sequence Length Molar Mass/kDa

p110α Lys-802 1068 aa^∗ 110

p110γ Lys-833 1102 aa 110

hVps34 Lys-636 887 aa 100

mTOR Lys-2187 2549 aa 280

DNA-PK Lys-3752 4128 aa 450

ATM Lys-3016 3056 aa 350

ATR Lys-2589 2644 aa 300

∗Amino acids= aa

20 O O

O O

O O

O

O

H

(1)

Figure 3 Structure of wortmannin (1).

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Wortmannin is a potent inhibitor of p110α, but also inhibits PI3Ks from other classes as well as similar proteins such as DNA-dependent protein kinase, ATM, ATR, mTOR and phosphoinositide-4-kinases. It has not yet been tested in clinical studies because of its weak selectivity. In addition, it exhibits liver toxicity and is insoluble in water. The low therapeutic index is also a result of instability. Wortmannin’s short half-life is due to early ring cleavage at C-20. Covalent, irreversible bonds are then formed between the ring-opened C-20 of wortmannin and Lys⁸⁰² in the ATP binding site of p110α (Fig. 4).⁶

Because of the toxicity and instability of wortmannin, many derivatives have been synthesized. One possibility is to replace the lactone by the initial steroid ring A. The acetoxy group at C-11 and methyl group at C-13 are

O

Figure 4 Inhibitory effect of wortmannin at Lys⁸⁰²of p110α.

(2)

Figure 5 Structure of demethoxyviridin (2) and the substituted analogues (3) and (4).

deleted. Hence, the conjugated system of the furan and B ring is expanded with an aromatic C ring. At C-1, the methoxy-methyl group is replaced by a free hydroxyl group. The resulting compound is demethoxyviridin (2) (Fig. 5) and can be found in the fungus Nodulisporium hinnuleum as a natural source.⁷

Demethoxyviridin is as unstable as wortmannin, because of the same furan ring structure. However, the half maximum inhibitory concentration (IC50) is 12-fold lower than that of wortmannin (IC50 = 12 nM). Therefore, alterations were performed at C-1 and C-20 to improve the half-life. At C-1 an esterification with succinic acid lead to enhanced stability in buffer solution. However, in liquid culture, medium stability decreased to about half a minute, a time similar to the half-life of wortmannin. In addition, the IC50of esterified demethoxyviridin (3) is 1.7 times higher than that of its parent compound. The C-1 substitution thus neither showed increased activity against PI3Ks nor improved stability.

Additional alterations at C-20 of demethoxyviridin should lead to open-ring inhibitors of PI3Ks. A derivative formed by adding glycine by its N-terminus to the furan was investigated (4). Like wortmannin, ana-logues react with the moiety of Lys⁸⁰² of p110α (Fig. 6). This compound with glycine results in improved stability in the presence of other free amino acids in cell plasma; the half-life increases to 64 minutes. Similar to the effect observed with esterified demethoxyviridin, the IC50 of the C-20-altered molecule is 3.6 times higher than that of wortmannin. The substance is less potent, but can still interact with lysine in the enzyme.

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(5) R¹= - C(CH₃)₃ R²= -(CH₂)₂- OH (6) R¹= - (CH₂)₃-N R²= - CH₃

OH O

O O

O O

O

O

H

N R¹ R²

Figure 6 Structure of two wortmannin analogues with high therapeutic indices.

To conclude, the problem posed by wortmannin’s instability⁸ can be solved by using open-ring analogues. These modified structures do not lose their potency as PI3K inhibitors.⁹Inhibition is mediated by an exchange of the coupled amine for lysine in the ATP-binding site of the kinase. The toxicity of all ring-cleaved analogues is decreased in comparison to the parent substance, wortmannin, but there exists a wide range of different modifications that show the same inhibitory structure-activity relationship (SAR). By attachment of primary or secondary amines to C-20 the half-life in cells can be increased and reactions with free amines in plasma can be slowed. Esterase activity in cells deacetylates C-11. An analogue (5) with a tertiary butylethanolamine moiety was not a viable substrate for esterase.

The derivative with a free hydroxyl group at C-11 was 20 to 100-fold less potent than its acetoxy analogue. This finding reveals that the furan ring is not the only site on the molecule important for inhibition. The C-11 substitution must interact with the pocket of ATP-binding site as well.

Furthermore, primary amines are weak inhibitors, while secondary amines exhibit a 20-fold lower IC50.

This higher potency can be explained by the fact that secondary amines can be easily substituted by primary ones under physiological conditions, like amino acids of the inhibited proteins. The primary amines are more reactive than the secondary ones.

For nucleophilic attack of lysine, it is irrelevant whether the amine moieties are polar or aprotic. Furthermore, SAR demonstrates that cyclic amines are weak inhibitors in comparison to aliphatic ones.

Another important point is that diamines are significantly more potent than their monoamine-substituted analogs. In summary, substance (6) is a more potent inhibitor than wortmannin, demethoxyviridin or other ring-opened analogues. Substance (6) has an aliphatic nitrogen moiety and con-tains a diamine and an acetylated form of C-11. It inhibited PI3K pathways and tumor growth in PTEN-negative glioma cells. The inhibitory effect was 426-fold more selective for PI3K than mTOR.⁶

Another ring-opened wortmannin analogue (PX-866) contains a dial-lylamine substitutent on C-20 (7) (Fig. 7).

PX-866 displayed comparable stability in solution to other ring-cleaved substances (Table 2). It inhibited Akt phosphorylation and decreased cell motility. In mice, growth of ovarian and lung tumors was inhibited without cytotoxicity. Liver toxicity was 65 percent less than the toxicity of wortman-nin and renal toxicity disappeared. PX-866 completely blocked the growth of glioblastoma cell lines at a concentration of 10 nM and already 1nM of the compound showed a decrease in cell growth while higher concentrations of wortmannin are necessary to reach the same effects. Still, wortmannin was a better inhibitor of PIP3 (phosphatidylinositol (3, 4, 5)-trisphosphate)

(7) OH O

O O

O O

O

O

H

N

Figure 7 Structure of PX-866.

Natural Products from Terrestrial Microbial Organisms with Cytotoxic Cell Cycle Inhibitors 155 Table 2 Inhibition of P13Ks by Wortmannin and Related Compounds

Substance IC50 Plasma Stability

Wortmannin 12 nM No

Demethoxyviridin 1 nM No

esterified demethoxyviridin (substance 3) 20 nM No

esterified and open-ring demethoxyviridin (substance 4) 44 nM Yes

17-hydroxywortmannin 2.7 nM No

substance 5 1110 nM Yes

substance 6 6.4 nM Yes

PX-866 0.1 nM Yes

synthesis in PTEN-deficient cells than PX-866 was. For cell cycle regulation, however, lipid phosphorylation is less important than protein phosphory-lation. Akt phosphorylation was most inhibited by Wortmannin in the first two hours of treatment. In comparison, PX-866 is effective over a longer period due to its higher stability. This viridin analogue is cytostatic rather than cytotoxic. It has been tested in combination with the established agent, cisplatin, and supra-additive effects have been found against cancer cells.