Methionine aminopeptidase (MetAP) enzymes require a divalent metal ion such as Mn(II), Fe(II), Co(II), Ni(II), or Zn(II) for its removal of the N-terminal methionine from newly synthesized proteins, but it is not certain which of these ions is most important in vivo. Metalloform-selective MetAP inhibitors could be valuable for defining which metals are physiologically relevant for MetAP activation and could serve as leads for development of new therapeutic agents. We have screened a library of 43 736 small drug-like molecules against Escherichia coli MetAP and identified two groups of potent and highly metalloform-selective inhibitors of the Co(II)-form, and of the Mn(II)-form, of this enzyme. Compound 1 is 790-fold more selective for the Co(II)-form, while compound 4 is over 640-fold more potent toward the Mn(II)-form. The X-ray structure of a di-Mn(II) form of E. coli MetAP complexed with the Mn(II)-form-selective compound 4 was obtained, and it shows that the inhibitor interacts with both Mn(II) ions through the two oxygen atoms of its free carboxylate group. The preferential coordination of the hard (oxygen) donors to Mn(II) may contribute to its superb selectivity toward the Mn(II)-form.

Methionine aminopeptidase (MetAP) removes the amino-terminal methionine residue from newly synthesized proteins, and it is a target for the development of antibacterial and anticancer agents. Available x-ray structures of MetAP, as well as other metalloaminopeptidases, show an active site containing two adjacent divalent metal ions bridged by a water molecule or hydroxide ion. The predominance of dimetalated structures leads naturally to proposed mechanisms of catalysis involving both metal ions. However, kinetic studies indicate that in many cases, only a single metal ion is required for full activity. By limiting the amount of metal ion present during crystal growth, we have now obtained a crystal structure for a complex of Escherichia coli MetAP with norleucine phosphonate, a transition-state analog, and only a single Mn(II) ion bound at the active site in the position designated M1, and three related structures of the same complex that show the transition from the mono-Mn(II) form to the di-Mn(II) form. An unliganded structure was also solved. In view of the full kinetic competence of the monometalated MetAP, the much weaker binding constant for occupancy of the M2 site compared with the M1 site, and the newly determined structures, we propose a revised mechanism of peptide bond hydrolysis by E. coli MetAP. We also suggest that the crystallization of dimetalated forms of metallohydrolases may, in some cases, be a misleading experimental artifact, and caution must be taken when structures are generated to aid in elucidation of reaction mechanisms or to support structure-aided drug design efforts.metalloprotease ͉ protein structure ͉ enzyme inhibition ͉ drug discovery ͉ metal occupancy A ll newly synthesized proteins have an amino-terminal methionine residue corresponding to the start codon AUG. In a significant number of cases, this initiating methionine residue is removed, either co-or posttranslationally, by the enzyme methionine aminopeptidase (MetAP) (1). In bacteria, this enzyme is the product of a single gene, and it is absolutely essential for bacterial survival, as demonstrated by gene deletion experiments in Escherichia coli (2) and Salmonella typhimurium (3). The single but critically essential MetAP enzyme of bacteria thus stands out as an attractive target for the design of antibacterial agents (4). Eukaryotes, on the other hand, have two distinct MetAPs, types I and II, arising from different genes (5). The human type II MetAP is a target of the antiangiogenic compounds fumagillin, ovalicin, and TNP-470 (6-8). Bengamides inhibit both types of MetAP (9) and cause inhibition of the growth of several human tumor cell lines in vitro at low-nanomolar concentrations. Therefore, human MetAPs may also serve as targets for the development of new anticancer agents. Some small molecules that inhibit MetAPs potently in vitro are known, but they lack potent antibacterial (10-12) or antiangiogenic (13) activities. One reason for this failure may be that they do not penetrate the bacterial or mammalian cells to...

Impromidine (IMP) and arpromidine (ARP)-derived guanidines are more potent and efficacious guinea pig (gp) histamine H 2 -receptor (gpH 2 R) than human (h) H 2 R agonists and histamine H 1 -receptor (H 1 R) antagonists with preference for hH 1 R relative to gpH 1 R. We examined N G -acylated imidazolylpropylguanidines (AIPGs), which are less basic than guanidines, at hH 2 R, gpH 2 R, rat H 2 R (rH 2 R), hH 1 R, and gpH 1 R expressed in Sf9 cells as probes for ligand-specific receptor conformations. AIPGs were similarly potent H 2 R agonists as the corresponding guanidines IMP and ARP, respectively. Exchange of pyridyl in ARP against phenyl increased AIPG potency 10-fold, yielding the most potent agonists at the hH 2 R-G s␣ fusion protein and gpH 2 R-G s␣ identified so far. Some AIPGs were similarly potent and efficacious at hH 2 R-G s␣ and gpH 2 R-G s␣ . AIPGs stabilized the ternary complex in hH 2 R-G s␣ and gpH 2 R-G s␣ differently than the corresponding guanidines. Guanidines, AIPGs, and small H 2 R agonists exhibited distinct agonist properties at hH 2 R, gpH 2 R, and rH 2 R measuring adenylyl cyclase activity. In contrast to ARP and IMP, AIPGs were partial H 1 R agonists exhibiting higher efficacies at hH 1 R than at gpH 1 R. This is remarkable because, so far, all bulky H 1 R agonists exhibited higher efficacies at gpH 1 R than at hH 1 R. Collectively, our data suggest that AIPGs stabilize different active conformations in hH 2 R, gpH 2 R, and rH 2 R than guanidines and that, in contrast to guanidines, AIPGs are capable of stabilizing a partially active state of hH 1 R.HIS exerts its biological effects through the H 1 R, H 2 R, H 3 R, and H 4 R, respectively (Hill et al., 1997;Hough, 2001). We are particularly interested in the H 1 R and H 2 R (Klinker et al., 1996;Seifert et al., 2003;Dove et al., 2004). The H 1 R couples to G q -proteins mediating phospholipase C activation, and the H 2 R couples to G s -proteins mediating AC activation (Hill et al., 1997). In some systems, the H 2 R also couples to G q -proteins (Kü hn et al., 1996;Leopoldt et al., 1997). We established expression systems for the H 1 R and H 2 R in Sf9 insect cells (Houston et al., 2002). Sf9 cells can be cultured in large amounts and yield high GPCR expression levels. In Sf9 cell membranes, GPCR/G-protein coupling can be measured with high sensitivity using the steady-state GTPase activity. An advantage of the GTPase assay is that it assesses GPCR/ G-protein coupling at a proximal level, avoiding potential bias introduced by assessing more downstream events, such as effector activation or changes in gene expression. In the case of the H 1 R, coupling of the GPCR to insect cell G qproteins is determined using RGS proteins as signal enhancers for GTPase activity (Houston et al., 2002;Seifert et al., 2003). In the case of the H 2 R, fusion proteins of GPCR and mammalian G s␣ -proteins are used Wenzel-Seifert et al., 2001). GPCR-G s␣ fusion proteins ensure a defined 1:1 stoichiometry of the coupling partners and their efficient in...

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