Although ∼50% of all types of human cancers harbour wild-type TP53, this p53 tumour suppressor is often deactivated through a concerted action by its abnormally elevated suppressors, MDM2, MDMX or SIRT1. Here, we report a novel small molecule Inauhzin (INZ) that effectively reactivates p53 by inhibiting SIRT1 activity, promotes p53-dependent apoptosis of human cancer cells without causing apparently genotoxic stress. Moreover, INZ stabilizes p53 by increasing p53 acetylation and preventing MDM2-mediated ubiquitylation of p53 in cells, though not directly in vitro. Remarkably, INZ inhibits cell proliferation, induces senescence and tumour-specific apoptosis, and represses the growth of xenograft tumours derived from p53-harbouring H460 and HCT116 cells without causing apparent toxicity to normal tissues and the tumour-bearing SCID mice. Hence, our study unearths INZ as a novel anti-cancer therapeutic candidate that inhibits SIRT1 activity and activates p53.
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...
A series of pyridine-2-carboxylic acid derivatives were synthesized according to the leads from the screening, and potent inhibitors have been obtained by structural modification. They have shown submicromolar inhibition of the enzymes (for example, for 9n, IC(50) = 130 nM for EcMetAP1 and IC(50) = 380 nM for ScMetAP1). They represent small-molecule MetAP inhibitors with novel structures different from alkylating fumagillin derivatives and peptidic bestatin-based MetAP inhibitor.
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