Nanette L. S. Que

Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2

Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90β, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralog-specific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers.

Accumulation of a Lipid A Precursor Lacking the 4*-Phosphate following Inactivation of the Escherichia coli lpxK Gene*

The lpxK gene has been proposed to encode the lipid A 4′-kinase in Escherichia coli (Garrett, T. A., Kadrmas, J. L., and Raetz, C. R. H. (1997)J. Biol. Chem. 272, 21855–21864). In cell extracts, the kinase phosphorylates the 4′-position of a tetraacyldisaccharide 1-phosphate precursor (DS-1-P) of lipid A, but the enzyme has not yet been purified because of instability. lpxK is co-transcribed with an essential upstream gene, msbA, with strong homology to mammalian Mdr proteins and ABC transporters.msbA may be involved in the transport of newly made lipid A from the inner surface of the inner membrane to the outer membrane. Insertion of an Ω-chloramphenicol cassette into msbA also halts transcription of lpxK. We have now constructed a strain in which only the lpxK gene is inactivated by inserting a kanamycin cassette into the chromosomal copy oflpxK. This mutation is complemented at 30 °C by a hybrid plasmid with a temperature-sensitive origin of replication carryinglpxK +. When this strain (designated TG1/pTAG1) is grown at 44 °C, the plasmid bearing thelpxK + is lost, and the phenotype of anlpxK knock-out mutation is unmasked. The growth of TG1/pTAG1 was inhibited after several hours at 44 °C, consistent with lpxK being an essential gene. Furthermore, 4′-kinase activity in extracts made from these cells was barely detectable. In accordance with the proposed biosynthetic pathway for lipid A, DS-1-P (the 4′-kinase substrate) accumulated in TG1/pTAG1 cells grown at 44 °C. The DS-1-P from TG1/pTAG1 was isolated, and its structure was verified by 1H NMR spectroscopy. DS-1-P had not been isolated previously from bacterial cells. Its accumulation in TG1/pTAG1 provides additional support for the pathway of lipid A biosynthesis inE. coli. Homologs of lpxK are present in the genomes of other Gram-negative bacteria.

A Deacylase in Rhizobium leguminosarum Membranes That Cleaves the 3-O-Linked β-Hydroxymyristoyl Moiety of Lipid A Precursors

Lipid A from the nitrogen-fixing bacteriumRhizobium leguminosarum displays many structural differences compared with lipid A of Escherichia coli. R. leguminosarum lipid A lacks the usual 1- and 4′-phosphate groups but is derivatized with a galacturonic acid substituent at position 4′.R. leguminosarum lipid A often contains an aminogluconic acid moiety in place of the proximal glucosamine 1-phosphate unit. Striking differences also exist in the secondary acyl chains attached to E. coli versus R. leguminosarumlipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of laurate and myristate in R. leguminosarum. Recently, we have found that lipid A isolated by pH 4.5 hydrolysis ofR. leguminosarum cells is more heterogeneous than previously reported (Que, N. L. S., Basu, S. S., White, K. A., and Raetz, C. R. H. (1998) FASEB J.12, A1284 (abstr.)). Lipid A species lacking the 3-O-linked β-hydroxymyristoyl residue on the proximal unit contribute to this heterogeneity. We now describe a membrane-bound deacylase from R. leguminosarum that removes a single ester-linked β-hydroxymyristoyl moiety from some lipid A precursors, including lipid X, lipid IVA, and (3-deoxy-d-manno-octulosonic acid)2-lipid IVA. The enzyme does not cleaveE. coli lipid A or lipid A precursors containing an acyloxyacyl moiety on the distal glucosamine unit. The enzyme is not present in extracts of E. coli or Rhizobium meliloti, but it is readily demonstrable in membranes ofPseudomonas aeruginosa, which also contains a significant proportion of 3-O-deacylated lipid A species. Optimal reaction rates are seen between pH 5.5 and 6.5. The enzyme requires a nonionic detergent and divalent metal ions for activity. It cleaves the monosaccharide lipid X at about 5% the rate of lipid IVAand (3-deoxy-d-manno-octulosonic acid)2-lipid IVA. 1H NMR spectroscopy of the deacylase reaction product, generated with lipid IVA as the substrate, confirms unequivocally that the enzyme cleaves only the ester-linked β-hydroxymyristoyl residue at the 3-position of the glucosamine disaccharide.

Bypassing drug-resistance mechanisms of prostate cancer with small-molecules that target androgen receptor chromatin interactions

Human androgen receptor (AR) is a hormone-activated transcription factor that is an important drug target in the treatment of prostate cancer. Current small-molecule AR antagonists, such as enzalutamide, compete with androgens that bind to the steroid-binding pocket of the AR ligand–binding domain (LBD). In castration-resistant prostate cancer (CRPC), drug resistance can manifest through AR-LBD mutations that convert AR antagonists into agonists, or by expression of AR variants lacking the LBD. Such treatment resistance underscores the importance of novel ways of targeting the AR. Previously, we reported the development of a series of small molecules that were rationally designed to selectively target the AR DNA-binding domain (DBD) and, hence, to directly interfere with AR–DNA interactions. In the current work, we have confirmed that the lead AR DBD inhibitor indeed directly interacts with the AR-DBD and tested that substance across multiple clinically relevant CRPC cell lines. We have also performed a series of experiments that revealed that genome-wide chromatin binding of AR was dramatically impacted by the lead compound (although with lesser effect on AR variants). Collectively, these observations confirm the novel mechanism of antiandrogen action of the developed AR-DBD inhibitors, establishing proof of principle for targeting DBDs of nuclear receptors in endocrine cancers. Mol Cancer Ther; 16(10); 2281–91. ©2017 AACR.