(= 5 in all groups

(= 5 in all groups. highly selective for PfMetAP1b and did not exhibit significant cytotoxicity against primary human fibroblasts. Most importantly, XC11 inhibited the proliferation of strains 3D7 [chloroquine (CQ)-sensitive] and Dd2 (multidrug-resistant) and is active in mouse malaria models for both CQ-sensitive and CQ-resistant strains. These results suggest that PfMetAP1b is a Cdh5 promising target and XC11 is an important lead compound for the development of novel antimalarial drugs. in culture (10), likely through inhibition of the malaria MetAP2 enzyme. Together, these observations raised the possibility that inhibition of other MetAP isoforms KB130015 may be sufficient to block malaria growth. In this study, we cloned all four isoforms of MetAP cDNA and obtained purified enzymes KB130015 with enzymatic activity for PfMetAP1a, b, and c, but not PfMetAP2. Using PfMetAP1b as a target, a high-throughput screen of a large chemical library led to a previously undescribed structural class of inhibitors for the enzyme. Structure/activity studies identified a potent inhibitor, XC11, that was highly selective for PfMetAP1b among the four malaria MetAP enzymes. XC11 and some other analogs blocked growth in KB130015 cell culture. Importantly, XC11 also inhibited both CQ-sensitive and -resistant mouse malaria strains, dramatically prolonging the survival of malaria-infected animals. These results suggest that selective targeting of PfMetAP1b is a promising strategy for the development of novel antimalarial drugs. Results Identification of PfMetAP1b as an Active Methionine Aminopeptidase Encoded in the Genome. We searched for genes that were homologous to the catalytic domains of human and yeast genes in the 3D7 genome database (http://plasmodb.org). Among the four putative genes, one was identified as (Gene ID: PF14_0327) based on the presence of the unique 64-aa insertion toward the C terminus of the catalytic domain. The remaining three showed high homology to MetAP1 from both human and yeast (Fig. 1) and were tentatively named (Gene ID: PFE1360c, PF10_0150, and MAL8P1.140, respectively). Similar to human and yeast MetAP1, all three putative PfMetAP1 proteins contained five highly conserved residues, two Asp, one His, and two Glu, that coordinate two metal ions to form the binuclear active sites of all MetAP enzymes known to date (Fig. 1). Of the three putative PfMetAP1 proteins, PfMetAP1b was most closely related to the human and yeast MetAP1 based on the zinc-finger motif present in its N-terminal extension, suggesting that PfMetAP1b may play an important role in malaria growth and survival. Open in a separate window Fig. 1. Protein sequence multialignment (ClustalW; www.ebi.ac.uk) for PfMetAP1a, PfMetAP1b, PfMetAP1c, Human MetAP1 (HuMetAP1), and Yeast MetAP1 (ScMetAP1). Their C-terminal catalytic domains were highly conserved, including the 5 metal-chelating residues (2Asp, 2Glu, 1His, highlighted in pink) coordinating two adjacent divalent metal ions. PfMetAP1a has no N-terminal extension. PfMetAP1b has zinc finger motif (highlighted in green) followed by a linker to C-terminal catalytic domain. PfMetAP1c has a signal peptide (highlighted in blue) followed by a transit peptide domain (highlighted in red) targeting the apicoplast (11) (as predicted by PlasmoAP, an online software from http://plasmodb.org) at the N-terminal. The C-terminal catalytic domain has an insertion (highlighted in brown) of 210 aa KB130015 inside, which is common in malaria protein (12). The full-length cDNA, amplified from a cDNA library, was subcloned into the pGEX-6P-2 vector, overexpressed in with a of 13.9 min?1, comparable with recombinant MetAP1 enzymes from other organisms (13, 14). The availability of large quantities of active recombinant PfMetAP1b protein and the convenient spectrophotometric enzymatic assay (13) enabled a high-throughput screening of library of compounds for PfMetAP1b inhibitors. Open in a separate window Fig. 2. Isolation of recombinant PfMetAP1b, structure of XC11, and assessment of its selectivity for PfMetAP1b among four putative PfMetAPs. (values in the enzymatic assay. The most potent hits of each structural class then were tested for their selectivity for PfMetAP1b among the four PfMetAP proteins as well as human MetAP enzymes and their ability to inhibit the growth KB130015 of in erythrocyte culture. From these follow-up analyses, one structural class, that which contained a 2-(2-pyridinyl)-pyrimidine core, stood out as the most promising inhibitors of PfMetAP1b and for PfMetAP1b inhibition by 30-fold. Of all of the analogs tested, XC11 emerged as the most potent inhibitor of PfMetAP1b, which was selected for further characterization (Fig. 2or insect cells (X.C. and J.O.L., unpublished data), we used a mammalian three-hybrid assay similar to the yeast three-hybrid system (15) to determine the potential interaction between XC11 and PfMetAP2. Two fusion proteins were coexpressed in 293T cells, one being the full-length PfMetAP2 fused with the VP16 transactivation domain and the other being the hormone-binding domain of rat glucocorticoid receptor fused to the Gal4.