P-Site Ligands

An early observation that 2 -d-3 -AMP was a more potent inhibitor of adenylyl cyclases than 2 -d-Ado suggested that the enzyme would accept substitutions at the 3 -ribose position and that phosphate was particularly well tolerated. This led to the generation of a family of 3 -phosphoryl derivatives of 2, 5 -dideoxyadenosine exhibiting ever greater inhibition with the addition of an increasing number of 3 -phosphoryl groups, the most potent of which is 2, 5 -dideoxyadenosine-3 -tetraphosphate (2, 5 -dd-3 -A4P Table 4) [5]. These constitute a class of inhibitors historically referred to as P -site ligands that caused inhibition of adenylyl... 

P-site ligands inhibit adenylyl cyclases by a noncompetitive, dead-end- (post-transition-state) mechanism (cf. Fig. 6). Typically this is observed when reactions are conducted with Mn2+ or Mg2+ on forskolin- or hormone-activated adenylyl cyclases. However, under- some circumstances, uncompetitive inhibition has been noted. This is typically observed with enzyme that has been stably activated with GTPyS, with Mg2+ as cation. That this is the mechanism of P-site inhibition was most clearly demonstrated with expressed chimeric adenylyl cyclase studied by the reverse reaction. Under these conditions, inhibition by 2 -d-3 -AMP was competitive with cAMP. That is, the P-site is not a site per se, but rather an enzyme configuration and these ligands bind to the post-transition-state configuration from which product has left, but before the enzyme cycles to accept new substrate. Consequently, as post-transition-state inhibitors, P-site ligands are remarkably potent and specific inhibitors of adenylyl cyclases and have been used in many studies of tissue and cell function to suppress cAMP formation. 

Fig. 15.4 P rinciple of second-site screening using a spin-labeled first ligand. First-site ligand 2 was spin-labeled to yield 2. The quenching effects of 2 on the resonances of any second-site ligand are observed.

However, the complex [Pd(dppomf)(COCH3)]+ undergoes immediate methanolysis at room temperature and even though (1) the dppomf acts as a tridentate 3-P, P, Fe ligand, with trans P atoms, and (2) ethene does not insert, which is indicative that a coordination site is not easily available. Thus, in this case, methanolysis either occurs via a five-coordinate transition state or it can occur also intermolecularly [47]. This might be also the case for the methoxycarbonylation of ethene catalysed by the osmocene analogue of dppf (Sect. 3.3.2) [79]. 

Asymmetric hydroboration of styrenes employing diphosphine complexes provides a successful solution to the generation of chirality at a benTyhc site, a potentially important route to many bioactive molecules. Since substitution on the double bond leads to severe loss of enantioselectivity, the apphcations are necessarily hmited. It was adventitiously discovered that the P,N-ligand QUINAP [47], which is effective in asymmetric... 

Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D., and Chambon, P. (1996) ligand-activated site-specific recombination in mice. Proc. Natl. Acad. Sci. USA 93, 10887-10890. 

Fig. 7 The location on tubulin of residues that modulate the sensitivity to MT-destabilizing agents and the location of exogenous inhibitor and nucleotide sites on P tubulin. The a subunit is in semitransparent pink together with a composite P-subunit color-coded as in Fig. 3a with ball-and-stick models of bound taxol (orange), colchicine (yellow) and GDP (magenta). Ball-and-stick models of vinblastine (cyan) are drawn on the two partial vinca sites on a and on P tubulin. The sulfur atom of Cys P12 is highlighted as a yellow sphere. The sites of nine amino acid substitutions [49] that both confer resistance to vinblastine and colchicine and stabilize MTs are depicted as red (on a tubulin) or green (on P tubulin) spheres. Two residues of the P H10 helix whose mutations enhance the sensitivity to colchicine site ligands and destabilize MTs [71] are also shown as blue spheres...

Colchicine site ligands are structurally simpler than taxol or vinblastine site compounds the extent of the colchicine site is also smaller. This is confirmed with the observation that podophyllotoxin interacts with the same P tubulin residues as colchicine [15]. Modehng studies also suggest a pocket of restricted size for other colchicine site ligands [40] while some extensions of the site have been postulated in an approach combining modeling and virtual screening [61]. Biochemical evaluations have confirmed some of the hits as new tubulin inhibitors but no structural data are available yet that confirm whether they actually make use of an additional cavity near the colchicine site. 

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