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Target Phosphatases for Drug Development

For "rational drug development," one of the first steps is the identification of suitable cellular targets, and to date most targets are proteins. In the development of antitumor agents, many protein kinases have been designated as developmental drug targets based on the roles they play in the regulation of signaling pathways known to affect cellular proliferation. However, for the serine/threonine phosphatases the process has been quite the opposite. That is, compounds with antitumor activity were identified first via conventional methods (i.e., the screening of natural product extracts). The realization that these antitumor drugs acted on protein phosphatases came much later, when investigations into the molecular mechanisms leading to the suppression of tumor progression revealed that the antitumor drugs acted as potent inhibitors of protein phosphatases.

To understand how phosphatase inhibitors with antitumor activity were discovered and developed, it will be helpful to first briefly review the enzymes upon which they act. Traditionally, serine/threonine phosphatases have been placed into four major groups (PP1, PP2A, PP2B, and PP2C) based on their biochemical properties. These enzymes have also been divided into two major gene families [protein phosphatase P (PPP) and protein phosphatase M (PPM)] based on the similarity of their primary amino acid sequence (Cohen 1997). The PPP gene family consists of PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7. It should also be noted that in humans there are actually three genes encoding four isoforms of PP1, two gene encoding iso-forms of PP2A, two genes encoding isoforms of PP7, and three genes encoding at least six isoforms of PP2B, with the additional isoforms produced by alternative splicing of mRNA (Honkanen and Golden 2002).

The catalytic subunits of all PPP-family members are structurally similar (Fig. 1). Nonetheless, they can be further classified by their sensitivity to a number of natural compounds that inhibit catalytic activity [i.e., okadaic acid, calyculin A, fostriecin, microcystin, cantharidin, and tautomycin (for review see Honkanen and Golden 2002; Lewy et al. 2002). PP1-PP6 are sensitive to inhibition and contain a similar "toxin"-binding domain near the C-terminal region of the catalytic subunit. In contrast, PP2B and PP7 have regions within the inhibitor-binding domains that are not conserved, and these "inserts" render them less sensitive, or insensitive, to the above listed inhibitors. The PPM family of enzymes, which is not affected by the above-mentioned inhibitors, consists of the Mg2+/Mn2+-dependent phosphatases (biochemically classified as PP2C) and the pyruvate dehydrogenase phos-phatases (Cohen 1997).

Fig. 1 Homology of serine/threonine protein phosphatases. A schematic representation depicting the amino acid similarity of human PPP-family serine/threonine protein phosphatases. PP1-PP7 contain a common catalytic core domain that is highly conserved. PP1, PP2A, PP4, and PP6 are highly homologous enzymes, differing primarily in their C-and N-terminal regions. PP2B differs in that it contains a Ca2+-calmodulin (CaM)-bind-ing domain in its C-terminal region and two small divergent regions (indicated by arrows and open boxes) in the catalytic domain near the okadaic acid/microcystin binding site. The amino-terminal region of PP5 contains three tetratricopeptide (TPR) domains. PP7 differs from all of the other PPase families in that it contains EF-hand motifs in the C-terminal region (indicated by filled squares) and a larger (44 amino acid) insert in the catalytic core domain (indicated by an open box). The inhibitor-insensitive enzymes contain divergent regions (inserts) in the inhibitor binding domain, which have been designated as the b12-b13 loop based on the crystal structure of PP1

Fig. 1 Homology of serine/threonine protein phosphatases. A schematic representation depicting the amino acid similarity of human PPP-family serine/threonine protein phosphatases. PP1-PP7 contain a common catalytic core domain that is highly conserved. PP1, PP2A, PP4, and PP6 are highly homologous enzymes, differing primarily in their C-and N-terminal regions. PP2B differs in that it contains a Ca2+-calmodulin (CaM)-bind-ing domain in its C-terminal region and two small divergent regions (indicated by arrows and open boxes) in the catalytic domain near the okadaic acid/microcystin binding site. The amino-terminal region of PP5 contains three tetratricopeptide (TPR) domains. PP7 differs from all of the other PPase families in that it contains EF-hand motifs in the C-terminal region (indicated by filled squares) and a larger (44 amino acid) insert in the catalytic core domain (indicated by an open box). The inhibitor-insensitive enzymes contain divergent regions (inserts) in the inhibitor binding domain, which have been designated as the b12-b13 loop based on the crystal structure of PP1

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