Stress response pathways allow cells to sense and respond to environmental changes and adverse pathophysiological states. Pharmacological modulation of cellular stress pathways has implications in the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. The quinone methide triterpene celastrol, derived from a traditional Chinese medicinal herb, has numerous pharmacological properties, and it is a potent activator of the mammalian heat shock transcription factor HSF1. However, its mode of action and spectrum of cellular targets are poorly understood. We show here that celastrol activates Hsf1 in Saccharomyces cerevisiae at a similar effective concentration seen in mammalian cells. Transcriptional profiling revealed that celastrol treatment induces a battery of oxidant defense genes in addition to heat shock genes. Celastrol activated the yeast Yap1 oxidant defense transcription factor via the carboxy-terminal redox center that responds to electrophilic compounds. Antioxidant response genes were likewise induced in mammalian cells, demonstrating that the activation of two major cell stress pathways by celastrol is conserved. We report that celastrol's biological effects, including inhibition of glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism for biological activity based on modification of key reactive thiols by this natural product. INTRODUCTIONThe heat shock response (HSR) is an ancient and highly conserved cytoprotective mechanism. Production of heat shock proteins, including protein chaperones, is essential for the folding, repair, or triage of damaged proteins; thus, it serves to promote cellular viability under conditions that would otherwise induce apoptosis (McMillan et al., 1998;Christians et al., 2002). There is significant interest in the discovery and development of small molecules that modulate the HSR and parallel stress response pathways for therapeutic purposes (Morimoto and Santoro, 1998;Westerheide and Morimoto, 2005;Corson and Crews, 2007). The HSR is governed by the stress-inducible heat shock transcription factor HSF1, which plays a key regulatory role in response to environmental stress, development, and many pathophysiological conditions, including cancer, ischemia-reperfusion injury, diabetes, and aging (Morimoto and Santoro, 1998;Morano and Thiele, 1999a).Significant progress has been made in identifying HSF1-modulating compounds such as triptolide and quercetin, inhibitors of HSF1, and activators such as the protein synthesis inhibitor puromycin, the proteasome inhibitor MG132, the nonsteroidal anti-inflammatory drugs salicylate and indomethacin, and the heat shock protein (Hsp)90 inhibitors radicicol and geldanamycin (Hightower, 1980;Jurivich et al., 1992;Lee et al., 1995;Nagai et al., 1995;Bagatell et al., 2000;Holmberg et al., 2000;Westerheide et al., 2006). A large-scale screen for novel pharmacologically active compounds that may be beneficial in treating the neur...

The natural product celastrol (1) possesses numerous beneficial therapeutic properties and affects numerous cellular pathways. The mechanism of action and cellular target(s) of celastrol, however, remain unresolved. While a number of studies have proposed that the activity of celastrol is mediated through reaction with cysteine residues, these observations have been based on studies with specific proteins or by in vitro analysis of a small fraction of the proteome. In this study, we have investigated the spatial and structural requirements of celastrol for the design of suitable affinity probes to identify cellular binding partners of celastrol. Although celastrol has several potential sites for modification, some of these were not synthetically amenable or yielded unstable analogs. Conversion of the carboxylic acid functionality to amides and long-chain analogs, however, yielded bioactive compounds that induced the heat shock response (HSR) and antioxidant response and inhibited Hsp90 activity. This led to the synthesis of biotinylated celastrols (23 and 24) that were used as affinity reagents in extracts of human Panc-1 cells to identify Annexin II, eEF1A, and β-tubulin as potential targets of celastrol.

Celastrol, an important natural product and Hsp90 inhibitor with a wide range of biological and medical activities and broad use as a biological probe, acts by an as yet undetermined mode of action. It is known to undergo Michael additions with biological sulfur nucleophiles. Here it is demonstrated that nucleophiles add to the pharmacophore of celastrol in a remarkable stereospecific manner. Extensive characterization of the addition products have been obtained using NMR spectrometry, nuclear Overhauser effects, and density functional theory to determine facial selectivity and gain insight into the orbital interactions of the reactive centers. This stereospecificity of celastrol may be important to its protein target selectivity.

Peptidoglycan (PG) is unique to bacteria, and thus, the enzymes responsible for its biosynthesis are promising antibacterial drug targets. The membrane-embedded enzymes in PG remain significant challenges in studying their mechanisms due to the fact that preparations of suitable enzymatic substrates require time-consuming biological transformations or chemical synthesis. Lipid I (prenyl diphosphoryl-MurNAc-pentapeptide) is an important PG biosynthesis intermediate to study the central enzymes, translocase I (MraY/MurX) and MurG. Lipid I isolated from nature contains the C50-or C55-prenyl unit that shows extremely poor water-solubility that renders studies of translocase I and MurG enzymes difficult. We have studied biological transformation of water soluble lipid I fluorescent probes using bacterial membrane fractions and purified MraY enzymes. In our investigation of the minimum structural requirements of the prenyl phosphates in MraY-catalyzed lipid I synthesis, we found that (2Z,6E)-farnesyl phosphate (C15-phosphate) can be recognized by E. coli MraY to generate the water-soluble lipid I fluorescent probes in high-yield. Under the optimized conditions, the same reaction was performed by using the purified MraY from Hydrogenivirga spp. to afford the lipid I analog with high-yield in a short reaction time.

The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 μM concentration.

The historically important phage ΦX174 kills its host bacteria by encoding a 91-residue protein antibiotic called protein E. Using single-particle electron cryo–microscopy, we demonstrate that protein E bridges two bacterial proteins to form the transmembrane YES complex [MraY, protein E, sensitivity to lysis D (SlyD)]. Protein E inhibits peptidoglycan biosynthesis by obstructing the MraY active site leading to loss of lipid I production. We experimentally validate this result for two different viral species, providing a clear model for bacterial lysis and unifying previous experimental data. Additionally, we characterize the Escherichia coli MraY structure—revealing features of this essential enzyme—and the structure of the chaperone SlyD bound to a protein. Our structures provide insights into the mechanism of phage-mediated lysis and for structure-based design of phage therapeutics.

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Potential formation and reactions of adamantane‐1,3‐dicarbenes 1–3 generated under different conditions and from different precursors, such as sodium salt of adamantane‐1,3‐dicarbaldehyde ditosylhydrazone (4a), sodium salt of 1,3‐diacetyladamantane ditosylhydrazone (5a), sodium salt of 1,3‐dibenzoyladamantane ditosylhydrazone (6a), and 1,3‐bis(diazobenzyl)adamantane (7) are reported. Carbene species generated thermally from 4a yielded bishomoadamantane (15), as a final product, via intramolecular insertion into adjacent CC bond and formation of putative anti‐Bredt olefin species, followed by hydrogen abstraction. Pyrolysis of the same sodium salt 4a in the presence of hydrogen donor n‐Bu3SnH afforded 1,3‐dimethyladamantane (17). Thermal decomposition of sodium salt 5a afforded 1,3‐divinyladamantane (14). However, thermal decomposition of sodium salt 6a and diazo‐precursor 7 gave benzonitrile as a sole identified product. On the contrary, photolysis of 7 afforded dimeric azine 21. Finally, the synthetic pathways of novel tosylhydrazone derivatives 4, 5, 6 and their corresponding sodium salts, as well as bis‐diazocompound 7 are described. Copyright © 2008 John Wiley & Sons, Ltd.