A Target Repurposing Approach Identifies N-myristoyltransferase as a New Candidate Drug Target in Filarial Nematodes

Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC₅₀ values of 2.5–10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis.     

Repurposing Hsp90 inhibitors as antibiotics targeting histidine kinases

To address the growing need for new antimicrobial agents, we explored whether inhibition of bacterial signaling machinery could inhibit bacterial growth. Because bacteria rely on two-component signaling systems to respond to environmental changes, and because these systems are both highly conserved and mediated by histidine kinases, inhibiting histidine kinases may provide broad spectrum antimicrobial activity. The histidine kinase ATP binding domain is conserved with the ATPase domain of eukaryotic Hsp90 molecular chaperones. To find a chemical scaffold for compounds that target histidine kinases, we leveraged this conservation. We screened ATP competitive Hsp90 inhibitors against CckA, an essential histidine kinase in Caulobacter crescentus that controls cell growth, and showed that the diaryl pyrazole is a promising scaffold for histidine kinase inhibition. We synthesized a panel of derivatives and found that they inhibit the histidine kinases C. crescentus CckA and Salmonella PhoQ but not C. crescentus DivJ; and they inhibit bacterial growth in both Gram-negative and Gram-positive bacterial strains.     

Hsp90 Inhibitors Exhibit Resistance-Free Antiviral Activity against Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is a major cause of respiratory illness in young children, leading to significant morbidity and mortality worldwide. Despite its medical importance, no vaccine or effective therapeutic interventions are currently available. Therefore, there is a pressing need to identify novel antiviral drugs to combat RSV infections. Hsp90, a cellular protein-folding factor, has been shown to play an important role in the replication of numerous viruses. We here demonstrate that RSV requires Hsp90 for replication. Mechanistic studies reveal that inhibition of Hsp90 during RSV infection leads to the degradation of a viral protein similar in size to the RSV L protein, the viral RNA-dependent RNA polymerase, implicating it as an Hsp90 client protein. Accordingly, Hsp90 inhibitors exhibit antiviral activity against laboratory and clinical isolates of RSV in both immortalized as well as primary differentiated airway epithelial cells. Interestingly, we find a high barrier to the emergence of drug resistance to Hsp90 inhibitors, as extensive growth of RSV under conditions of Hsp90 inhibition did not yield mutants with reduced sensitivity to these drugs. Our results suggest that Hsp90 inhibitors may present attractive antiviral therapeutics for treatment of RSV infections and highlight the potential of chaperone inhibitors as antivirals exhibiting high barriers to development of drug resistance.     

The 90-kDa Heat Shock Protein Hsp90 Protects Tubulin against Thermal Denaturation

Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.     

Repurposing Pan-HDAC Inhibitors for ARID1A-Mutated Ovarian Cancer

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A fluorescence polarization assay for inhibitors of Hsp90

Hsp90 encodes a ubiquitous molecular chaperone protein conserved among species which acts on multiple substrates, many of which are important cell-signaling proteins. Inhibition of Hsp90 function has been promoted as a mechanism to degrade client proteins involved in tumorigenesis and disease progression. Several assays to monitor inhibition of Hsp90 function currently exist but are limited in their use for a drug discovery campaign. Using data from the crystal structure of an initial hit compound, we have developed a fluorescence polarization assay to monitor binding of compounds to the ATP-binding site of Hsp90. This assay is very robust (Z' > 0.9) and can detect affinity of compounds with IC50s to 40 nM. We have used this assay in conjunction with cocrystal structures of small molecules to drive a structure-based design program aimed at the discovery and optimization of a novel class of potent Hsp90 inhibitors.     

A comparison of Hsp90alpha and Hsp90beta interactions with cochaperones and substrates.

Hsp90 chaperone complexes function in assembly, folding, and activation of numerous substrates. The 2 vertebrate homologues encoded by the genes hsp90a and hsp90b are differentially expressed in embryonic and adult tissues and during stress; however, it is not known whether they possess identical functional activities in chaperone complexes. This question was addressed by examining potential differences between the Hsp90 isoforms with respect to both cochaperone and substrate interactions. Epitope-tagged proteins were expressed in mammalian cells or Xenopus oocytes and subjected to immunoprecipitation with an array of cochaperones. Both isoforms were shown to participate equally in multichaperone complexes, and no significant differences in cochaperone distribution were observed. The substrates Raf-1, HSF1, Cdc37, and MEK1 interacted with both Hsp90alpha and Hsp90beta, and the relative patterns of these interactions were not affected by heat shock. The substrate kinases c-Src, CKIIB, A-raf, and Erk interacted with both isoforms; however, significantly more Hsp90alpha was recovered after heat shock. The data demonstrate that Hsp90alpha and Hsp90beta exhibit similar interactions with cochaperones, but significantly different behaviors with respect to substrate interactions under stress conditions. These results reveal both functional similarities and key functional differences in the individual members of this protein family.     

A Grp on the Hsp90 mechanism

In a recent issue of Molecular Cell, Dollins et al. (2007) present the crystal structure of Grp94, which highlights the similarity between Grp94 and Hsp90 and provides insight into the resting state of Grp94 and potentially other Hsp90 family members.     

Identification,Design and Bio-Evaluation of Novel Hsp90 Inhibitors by Ligand-Based Virtual Screening

Heat shock protein 90 (Hsp90), whose inhibitors have shown promising activity in clinical trials, is an attractive anticancer target. In this work, we first explored the significant pharmacophore features needed for Hsp90 inhibitors by generating a 3D-QSAR pharmacophore model. It was then used to virtually screen the SPECS databases, identifying 17 hits. Compound S1 and S13 exhibited the most potent inhibitory activity against Hsp90, with IC₅₀ value 1.61±0.28 μM and 2.83±0.67 μM, respectively. Binding patterns analysis of the two compounds with Hsp90 revealed reasonable interaction modes. Further evaluation showed that the compounds exhibited good anti-proliferative effects against a series of cancer cell lines with high expression level of Hsp90. Meanwhile, S13 induced cell apoptosis in a dose-dependent manner in different cell lines. Based on the consideration of binding affinities, physicochemical properties and toxicities, 24 derivatives of S13 were designed, leading to the more promising compound S40, which deserves further optimization.     

A systematic protocol for the characterization of Hsp90 modulators

Several Hsp90 modulators have been identified including the N-terminal ligand geldanamycin (GDA), the C-terminal ligand novobiocin (NB), and the co-chaperone disruptor celastrol. Other Hsp90 modulators elicit a mechanism of action that remains unknown. For example, the natural product gedunin and the synthetic anti-spermatogenic agent H2-gamendazole, recently identified Hsp90 modulators, manifest biological activity through undefined mechanisms. Herein, we report a series of biochemical techniques used to classify such modulators into identifiable categories. Such studies provided evidence that gedunin and H2-gamendazole both modulate Hsp90 via a mechanism similar to celastrol, and unlike NB or GDA.     

A role for Hsp90 in cell cycle control: Wee1 tyrosine kinase activity requires interaction with Hsp90.

Wee1 protein kinase regulates the length of G2 phase by carrying out the inhibitory tyrosyl phosphorylation of Cdc2-cyclin B kinase. Mutations were isolated that suppressed the G2 cell cycle arrest caused by overproduction of Wee1. One class of swo (suppressor of wee1 overproduction) mutation, exemplified by swo1-26, also caused a temperature sensitive lethal phenotype in a wee1+ background. The swo1+ gene encodes a member of the Hsp90 family of stress proteins. Swo1 is essential for viability at all temperatures. Swo1 coimmunoprecipitates with Wee1, showing that the two proteins interact. The swo1-26 mutant undergoes premature mitosis when grown at a semi-permissive temperature. These data strongly indicate that formation of active Wee1 tyrosine kinase requires interaction with Swo1, perhaps in a manner analogous to the previously demonstrated interaction between Hsp90 and v-src tyrosine kinase. These observations demonstrate a unexpected role for Hsp90 in cell cycle control.     

High-throughput screening for Hsp90 ATPase inhibitors

Recently, we reported a useful assay for the determination of yeast Hsp90 ATPase activity. Using this assay, high-throughput screening of approximately 10,000 compounds was performed to determine the feasibility of this assay on large scale. Results from high-throughput screening indicated that the assay was reproducible (av Z-factor = 0.80) and identified 0.57% of the compounds as Hsp90 inhibitors that exhibited IC50s less than 20 microM. The structures of several of these inhibitory scaffolds are reported along with their IC50 values.     

Defining the requirements for Hsp40 and Hsp70 in the Hsp90 chaperone pathway

The Hsp90 chaperoning pathway and its model client substrate, the progesterone receptor (PR), have been used extensively to study chaperone complex formation and maturation of a client substrate in a near native state. This chaperoning pathway can be reconstituted in vitro with the addition of five proteins plus ATP: Hsp40, Hsp70, Hop, Hsp90, and p23. The addition of these proteins is necessary to reconstitute hormone-binding capacity to the immuno-isolated PR. It was recently shown that the first step for the recognition of PR by this system is binding by Hsp40. We compared type I and type II Hsp40 proteins and created point mutations in Hsp40 and Hsp70 to understand the requirements for this first step. The type I proteins, Ydj1 and DjA1 (HDJ2), and a type II, DjB1 (HDJ1), act similarly in promoting hormone binding and Hsp70 association to PR, while having different binding characteristics to PR. Ydj1 and DjA1 bind tightly to PR whereas the binding of DjB1 apparently has rapid on and off rates and its binding cannot be observed by antibody pull-down methods using either purified proteins or cell lysates. Mutation studies indicate that client binding, interactions between Hsp40 and Hsp70, plus ATP hydrolysis by Hsp70 are all required to promote conformational maturation of PR via the Hsp90 pathway.     

High-throughput assay for the identification of Hsp90 inhibitors based on Hsp90-dependent refolding of firefly luciferase

Previously, we have demonstrated that the renaturation of heat denatured firefly luciferase is dependent upon the activity of Hsp90 in rabbit reticulocyte lysate. Here, we demonstrate that this assay may identify inhibitors that obstruct the chaperone activity of Hsp90 either by direct binding to its N-terminal or C-terminal nucleotide binding sites or by interference with the ability of the chaperone to switch conformations. The assay was adapted and optimized for high-throughput screening. Greater than 20,000 compounds were screened to demonstrate the feasibility of using this assay on a large scale. The assay was reproducible (av Z-factor=0.62) and identified 120 compounds that inhibited luciferase renaturation by greater than 70% at a concentration of 12.5 microg/mL. IC50 values for twenty compounds with varying structures were determined for inhibition of luciferase refolding and in cell-based assays for Hsp90 inhibition. Several compounds had IC50 values <10 microM and represent a number of new lead structures with the potential for further development and optimization as potent Hsp90 inhibitors.     

Approaches for defining the Hsp90-dependent proteome

Hsp90 is the target of ongoing drug discovery studies seeking new compounds to treat cancer, neurodegenerative diseases, and protein folding disorders. To better understand Hsp90's roles in cellular pathologies and in normal cells, numerous studies have utilized proteomics assays and related high-throughput tools to characterize its physical and functional protein partnerships. This review surveys these studies, and summarizes the strengths and limitations of the individual attacks. We also include downloadable spreadsheets compiling all of the Hsp90-interacting proteins identified in more than 23 studies. These tools include cross-references among gene aliases, human homologues of yeast Hsp90-interacting proteins, hyperlinks to database entries, summaries of canonical pathways that are enriched in the Hsp90 interactome, and additional bioinformatic annotations. In addition to summarizing Hsp90 proteomics studies performed to date and the insights they have provided, we identify gaps in our current understanding of Hsp90-mediated proteostasis. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).