Activation of p53 tumor suppressor by antagonizing its negative regulator murine double minute (MDM)2 has been considered an attractive strategy for cancer therapy and several classes of p53-MDM2 binding inhibitors have been developed. However, these compounds do not inhibit the p53-MDMX interaction, and their effectiveness can be compromised in tumors overexpressing MDMX. Here, we identify small molecules that potently block p53 binding with both MDM2 and MDMX by inhibitor-driven homo-and/or heterodimerization of MDM2 and MDMX proteins. Structural studies revealed that the inhibitors bind into and occlude the p53 pockets of MDM2 and MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This mode of action effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. Dual MDM2/MDMX antagonists restored p53 apoptotic activity in the presence of high levels of MDMX and may offer a more effective therapeutic modality for MDMXoverexpressing cancers.T he tumor suppressor p53 is a powerful growth-suppressive and proapoptotic protein tightly controlled by its negative regulators: murine double minute (MDM)2 and MDMX (1, 2). These proteins bind p53 with their structurally similar N-terminal domains and effectively inhibit p53 transcriptional activity (1, 3). They both possess a RING (really interesting new gene) domain in their C termini, but it is only functional in MDM2, which serves as a specific E3 ligase and main regulator of p53 stability (4, 5). Despite its RING domain, MDMX does not have an intrinsic ligase activity and does not affect directly p53 stability (6). However, MDMX can enhance ligase activity of MDM2 toward p53 by forming MDM2/MDMX heterodimers (7,8). It has been reported that the MDM2/MDMX complex is responsible for polyubiquitination of p53, whereas MDM2 alone primarily induces monoubiquitination (9). Targeted disruption of MDM2/MDMX heterocomplexes is embryonic-lethal in mice, suggesting that complex formation is essential for p53 regulation in vivo (10). On the other hand, MDM2 can also ubiquitinate MDMX and is, therefore, responsible for its stability as well (11,12). MDM2 is a transcriptional target of p53, and both proteins form an autoregulatory feedback loop by which they mutually control their cellular levels (13).The functional relationship between MDM2 and MDMX is still being refined at the molecular level, but it is well established that these two negative regulators play a critical role in controlling p53 tumor-suppressor function in normal cells (2,14). This is why they are frequently overproduced through gene amplification and/or overexpression in tumors that retain wildtype p53 (14). Therefore, antagonizing the binding of MDM2 and MDMX to p53 is expected to restore p53 function and may offer a strategy for cancer therapy (15). Recently identified small-molecule inhibitors of the p53-MDM2 interaction have validated this approach, and the first pharmacological MDM2 antagonists ar...

This digest covers some of the most relevant progress in malaria drug disco very published betwe en 2010 and 2012. There is an urgent need to develop new antimalarial drugs. Such drugs can target the blood stage of the disease to alleviate the symptoms, the liver stage to prevent relapses, and the transmission stage to protect other humans. The pipeline for the blood stage is becoming robust, but this should not be a source of complacency, as the current therapies set a high standard. Drug disco very efforts directed towards the liver and transmission stages are in their infancy but are receiving increasing attention as targeting these stages could be instrumental in eradicating malaria.

The lipoglycodepsipeptide antibiotic ramoplanin is proposed to inhibit bacterial cell wall biosynthesis by binding to intermediates along the pathway to mature peptidoglycan, which interferes with further enzymatic processing. Two sequential enzymatic steps can be blocked by ramoplanin, but there is no definitive information about whether one step is inhibited preferentially. Here we use inhibition kinetics and binding assays to assess whether ramoplanin and the related compound enduracidin have an intrinsic preference for one step over the other. Both ramoplanin and enduracidin preferentially inhibit the transglycosylation step of peptidoglycan biosynthesis compared with the MurG step. The basis for stronger inhibition is a greater affinity for the transglycosylase substrate Lipid II over the MurG substrate Lipid I. These results provide compelling evidence that ramoplanin's and enduracidin's primary cellular target is the transglycosylation step of peptidoglycan biosynthesis.

Full details of a convergent total synthesis of the ramoplanin A2 and ramoplanose aglycon are disclosed. Three key subunits composed of residues 3-9 (heptapeptide 15), pentadepsipeptide 26 (residues 1, 2 and 15-17), and pentapeptide 34 (residues 10-14) were prepared, sequentially coupled, and cyclized to provide the 49-membered depsipeptide core of the aglycon. Key to the preparation of the pentadepsipeptide 26 incorporating the backbone ester was the asymmetric synthesis of an orthogonally protected l-threo-beta-hydroxyasparagine and the development of effective and near-racemization free conditions for esterification of its hindered alcohol (EDCI, DMAP, 0 degrees C). The coupling sites were chosen to maximize the convergency of the synthesis including that of the three subunits, to prevent late stage racemization of carboxylate-activated phenylglycine-derived residues, and to enlist beta-sheet preorganization of an acyclic macrocyclization substrate for 49-membered ring closure. By altering the order of final couplings, two macrocyclization sites, Phe(9)-d-Orn(10) and Gly(14)-Leu(15), were examined. Macrocyclization at the highly successful Phe(9)-d-Orn(10) site (89%) may benefit from both beta-sheet preorganization as well as closure at a d-amine terminus within the confines of a beta-turn at the end of the H-bonded antiparallel beta-strands. A more modest, but acceptable macrocyclization reaction at the Gly(14)-Leu(15) site (40-50%) found at the other end of the H-bonded antiparallel beta-strands within a small flexible loop may also benefit from preorganization of the cyclization substrate, is conducted on a substrate incapable of competitive racemization, and accommodates the convergent preparation of analogues bearing depsipeptide modifications. Deliberate late-stage incorporation of the subunit bearing the labile depsipeptide ester and a final stage Asn(1) side-chain introduction provides future access to analogues of the aglycons which themselves are equally potent or more potent than the natural products in antimicrobial assays.

A convergent total synthesis of the ramoplanin A2 and ramoplanose aglycon is disclosed. Three key subunits composed of residues 3-9 (heptapeptide 15), pentadepsipeptide 26, and pentapeptide 34 (residues 10-14) were prepared, sequentially coupled, and cyclized to provide the 49-membered depsipeptide core of the aglycon. Key to the preparation of the pentadepsipeptide 26 incorporating the backbone ester was the asymmetric synthesis of an orthogonally protected L-threo-beta-hydroxyasparagine and the development of effective and near-racemization free conditions for esterification of its hindered alcohol (EDCI, DMAP, 0 degrees C). The coupling sites were chosen to maximize the convergency of the synthesis including that of the three subunits, to prevent late stage racemization of carboxylate-activated phenylglycine-derived residues, and to enlist beta-sheet preorganization of an acyclic macrocyclization substrate for 49-membered ring closure. As such, macrocyclization at the chosen Phe(9)-D-Orn(10) site may benefit from both beta-sheet preorganization as well as closure at a D-amine terminus. Deliberate late stage incorporation of the subunit bearing the labile depsipeptide ester and a final stage Asn(1) side chain introduction provides future access to analogues of the aglycons which themselves are reported to be equally potent or more potent than the natural products in antimicrobial assays.