Novobiocin Analogues Research Articles

Protection against Aβ-induced neuronal damage by KU-32: PDHK1 inhibition as important target.

A feature of most neurodegenerative diseases is the presence of "mis-folded proteins" that form aggregates, suggesting suboptimal activity of neuronal molecular chaperones. Heat shock protein 90 (Hsp90) is the master regulator of cell responses to "proteotoxic" stresses. Some Hsp90 modulators activate cascades leading to upregulation of additional chaperones. Novobiocin is a modulator at the C-terminal ATP-binding site of Hsp90. Of several novobiocin analogs synthesized and tested for protection against amyloid beta (Aβ)-induced neuronal death, "KU-32" was the most potent in protecting primary neurons, but did not increase expression of other chaperones believed to help clear misfolded proteins. However, KU-32 reversed Aβ-induced superoxide formation, activated Complex I of the electron transfer chain in mitochondria, and blocked the Aβ-induced inhibition of Complex I in neuroblastoma cells. A mechanism for these effects of KU-32 on mitochondrial metabolism appeared to be the inhibition of pyruvate dehydrogenase kinase (PDHK), both in isolated brain mitochondria and in SH-SY5Y cells. PDHK inhibition by the classic enzyme inhibitor, dichloroacetate, led to neuroprotection from Aβ25-35-induced cell injury similarly to KU-32. Inhibition of PDHK in neurons would lead to activation of the PDH complex, increased acetyl-CoA generation, stimulation of the tricarboxylic acid cycle and Complex I in the electron transfer chain, and enhanced oxidative phosphorylation. A focus of future studies may be on the potential value of PDHK as a target in AD therapy.

Antitumor activity of nanoliposomes encapsulating the novobiocin analog 6BrCaQ in a triple-negative breast cancer model in mice

In this study, we investigated the anticancer efficacy of pegylated liposomes containing 6BrCaQ, an hsp90 inhibitor derived from novobiocin. 6BrCaQ has been previously identified as the most potent compound in a series of quinoleic novobiocin analogs but is poorly water-soluble. We investigated, for the first time, the anti-proliferative effects of this drug in vivo in an orthotopic breast cancer model (MDA-MB-231 luc) using pegylated liposomes to allow its administration. Hsp90, hsp70 and hsp27 protein and mRNA expressions were not strongly affected after treatment meaning it did not induce a heat shock response often associated with resistance and poor prognosis. Liposomal delivery of 6BrCaQ retarded tumor growth at a low dose (1 mg/kg, injected once a week for 4 weeks). Histological analysis of tumors revealed necrosis and a lower proportion of proliferative cells in treated mice indicating that this drug has potential for breast cancer therapy when encapsulated in liposomes.

Novobiocin Enhances Polymyxin Activity by Stimulating Lipopolysaccharide Transport.

Gram-negative bacteria are challenging to kill with antibiotics due to their impenetrable outer membrane containing lipopolysaccharide (LPS). The polymyxins, including colistin, are the drugs of last resort for treating Gram-negative infections. These drugs bind LPS and disrupt the outer membrane; however, their toxicity limits their usefulness. Polymyxin has been shown to synergize with many antibiotics including novobiocin, which inhibits DNA gyrase, by facilitating transport of these antibiotics across the outer membrane. Recently, we have shown that novobiocin not only inhibits DNA gyrase but also binds and stimulates LptB, the ATPase that powers LPS transport. Here, we report the synthesis of novobiocin derivatives that separate these two activities. One analog retains LptB-stimulatory activity but is unable to inhibit DNA gyrase. This analog, which is not toxic on its own, nevertheless enhances the lethality of polymyxin by binding LptB and stimulating LPS transport. Therefore, LPS transport agonism contributes substantially to novobiocin-polymyxin synergy. We also report other novobiocin analogs that inhibit DNA gyrase better than or equal to novobiocin, but bind better to LptB and therefore have even greater LptB stimulatory activity. These compounds are more potent than novobiocin when used in combination with polymyxin. Novobiocin analogs optimized for both gyrase inhibition and LPS transport agonism may allow the use of lower doses of polymyxin, increasing its efficacy and safety.

Synthesis and Biological Evaluation of Stilbene Analogues as Hsp90 C-Terminal Inhibitors.

The design, synthesis, and biological evaluation of stilbene-based novobiocin analogues is reported. Replacement of the biaryl amide side chain with a triazole side chain produced compounds that exhibited good antiproliferative activities. Heat shock protein 90 (Hsp90) inhibition was observed when N-methylpiperidine was replaced with acyclic tertiary amines on the stilbene analogues that also contain a triazole-derived side chain. These studies revealed that ≈24 Å is the optimal length for compounds that exhibit good antiproliferative activity as a result of Hsp90 inhibition.

Novobiocin Analogs as Potential Anticancer Agents.

The aminocoumarin antibiotic, novobiocin, is a natural product that inhibits DNA gyrase, a bacterial enzyme involved in cell division. More recently, novobiocin was found to act also on eukaryotic cells by blocking the 90 kDa heat shock protein (Hsp90). Hsp90 is a molecular chaperone, critical for folding, stabilization and activation of many proteins, in particular oncoproteins responsible for cancer progression. As opposed to the geldanamycin and radicicol, the known inhibitors of Hsp90 that bind to the N-terminal region, the binding domain of novobiocin is localized in the C-terminal part of this protein. While the N-terminal inhibition also leads to the induction of some pro-survival signals, C-terminal inhibitors in which prosurvival responses are avoided and client degradation is maintained can be developed as a new class of potential anticancer chemotherapeutics. Numerous novobiocin analogs have been designed in the search for more potent compounds and some of them exhibit significantly enhanced anti-proliferative activity versus the natural product, as evaluated by cellular efficacies against several cancer cell lines. This review describes structure-activity-relationships of novobiocin analogs and some biological data reported so far on the anticancer activity of these modified compounds.

Ferrocenyl and organic novobiocin derivatives: Synthesis and their in vitro biological activity

A focused series of novobiocin derivatives containing a ferrocene unit together with their corresponding organic novobiocin analogues have been synthesized in modest to good yields. These compounds were screened for biological activity against a chloroquine-sensitive strain of Plasmodium falciparum (3D7) and human breast cancer cell line (HCC38). With the exception of compounds 5c and 5d, the general trend observed is that incorporation of the ferrocene moiety into novobiocin scaffold resulted in compounds 6a–d/6f showing enhanced activity compared to organic analogues 5a–b and 5e–f.

Diverging Novobiocin Anti-Cancer Activity from Neuroprotective Activity through Modification of the Amide Tail.

Novobiocin is a natural product that binds the Hsp90 C-terminus and manifests Hsp90 inhibitory activity. Structural investigations on novobiocin led to the development of both anti-cancer and neuroprotective agents. The varied pharmacological activity manifested by these novobiocin analogs prompted the investigation of structure-function studies to identify these contradictory effects, which revealed that modifications to the amide side chain produce either anti-cancer or neuroprotective activity. Compounds that exhibit neuroprotective activity contain a short alkyl or cycloalkyl amide side chain. In contrast, anti-cancer agents contain five or more carbons, disrupt interactions between Hsp90α and Aha1, and induce the degradation of Hsp90-dependent client proteins.

Novel 1,6-naphthyridin-2(1H)-ones as potential anticancer agents targeting Hsp90

Hsp90 is an ATP-dependent chaperone known to be overexpressed in many cancers. This way, Hsp90 is an important target for drug discovery. Novobiocin, an aminocoumarin antibiotic, was reported to inhibit Hsp90 targeting C-terminal domain, and showed anti-proliferative properties, leading to the development of new and more active compounds. Consequently, a new set of novobiocin analogs derived from 1,6-naphthyridin-2(1H)-one scaffold was designed, synthesized and evaluated against two breast cancer cell lines. Subsequently, cell cycle progression and apoptosis were conducted on best candidates, finally Western Blot analysis was performed to measure their ability to induce degradation of Hsp90 client proteins.

Novobiocin Analogues That Inhibit the MAPK Pathway.

Heat shock protein 90 (Hsp90) inhibition by modulation of its N- or C-terminal binding site has become an attractive strategy for the development of anticancer chemotherapeutics. The first Hsp90 C-terminus inhibitor, novobiocin, manifested a relatively high IC50 value of ∼700 μM. Therefore, investigation of the novobiocin scaffold has led to analogues with improved antiproliferative activity (nanomolar concentrations) against several cancer cell lines. During these studies, novobiocin analogues that do not inhibit Hsp90 were identified; however, these analogues demonstrated potent antiproliferative activity. Compound 2, a novobiocin analogue, was identified as a MAPK pathway signaling disruptor that lacked Hsp90 inhibitory activity. In addition, structural modifications of compound 2 were identified that segregated Hsp90 inhibition from MAPK signaling disruption. These studies indicate that compound 2 represents a novel scaffold for disruption of MAPK pathway signaling and may serve as a useful structure for the generation of new anticancer agents.

Modulating Molecular Chaperones Improves Mitochondrial Bioenergetics and Decreases the Inflammatory Transcriptome in Diabetic Sensory Neurons.

We have previously demonstrated that modulating molecular chaperones with KU-32, a novobiocin derivative, ameliorates physiologic and bioenergetic deficits of diabetic peripheral neuropathy (DPN). Replacing the coumarin core of KU-32 with a meta-fluorinated biphenyl ring system created KU-596, a novobiocin analogue (novologue) that showed neuroprotective activity in a cell-based assay. The current study sought to determine whether KU-596 offers similar therapeutic potential for treating DPN. Administration of 2-20 mg/kg of KU-596 improved diabetes induced hypoalgesia and sensory neuron bioenergetic deficits in a dose-dependent manner. However, the drug could not improve these neuropathic deficits in diabetic heat shock protein 70 knockout (Hsp70 KO) mice. To gain further insight into the mechanisms by which KU-596 improved DPN, we performed transcriptomic analysis of sensory neuron RNA obtained from diabetic wild-type and Hsp70 KO mice using RNA sequencing. Bioinformatic analysis of the differentially expressed genes indicated that diabetes strongly increased inflammatory pathways and that KU-596 therapy effectively reversed these increases independent of Hsp70. In contrast, the effects of KU-596 on decreasing the expression of genes regulating the production of reactive oxygen species were more Hsp70-dependent. These data indicate that modulation of molecular chaperones by novologue therapy offers an effective approach toward correcting nerve dysfunction in DPN but that normalization of inflammatory pathways alone by novologue therapy seems to be insufficient to reverse sensory deficits associated with insensate DPN.

Design, synthesis, and biological evaluation of ring-constrained novobiocin analogues as hsp90 C-terminal inhibitors.

Hsp90 C-terminal inhibitors represent a novel and alternative chemotherapeutic approach for the treatment of cancer. Novobiocin was the first natural product identified as an Hsp90 C-terminal inhibitor; however, it manifests poor antiproliferative activity. In contrast to N-terminal inhibitors, novobiocin does not induce the pro-survival heat shock response. Structural investigations on novobiocin have elucidated some structure-activity relationships and several promising compounds. On the basis of structure-activity relationships and computational studies, a library of ring-constrained novobiocin analogues was designed, synthesized, and evaluated in antiproliferative assays. Results obtained from these studies provide insights into the Hsp90 C-terminal binding site, and new analogues that were developed manifest low micromolar to mid-nanomolar antiproliferative activity resulting from Hsp90 inhibition.

Synthesis and antiproliferative activity of novobiocin analogues as potential hsp90 inhibitors

A series of substituted coumarins1–10 was designed and synthesized as a novel class of 4TCNA analogues. Compound 2a showed excellent antiproliferative activity with mean GI50 values at a micromolar level in a diverse set of human cancer cells (GI50 = 2–30 μM) and induced a high apoptosis level in MCF-7 breast cancer cell line. The molecular signature of hsp90 inhibition was assessed by depletion of the Erα hsp90 client protein.

Synthesis of 3-aminochromenes: the Zincke reaction revisited

3-Aminocoumarines and 2-iminochromen-3-amines were efficiently prepared from the Zincke-ring-opening reaction of the corresponding 2H-chromen-3-pyridinium chlorides using N-methylpiperazine. This methodology unravels the marked potential of pyridinium salts as protective groups for primary amines.These scaffolds can be considered important building blocks for new novobiocin analogues and heterocyclic compounds.

Novologues containing a benzamide side chain manifest anti-proliferative activity against two breast cancer cell lines

Hsp90 represents a promising target for the development of both anti-cancer and neuroprotective agents. Structure–activity relationship studies on novobiocin and novobiocin analogues, led to the development of KU-32 and recently, KU-596, as lead compounds for the potential treatment of neurodegenerative diseases. Similar to KU-32, we have demonstrated that upon replacement of the acetamide side chain present in KU-32 with a benzamide, this neuroprotective agent was transformed into a scaffold that manifests anti-proliferative activity. To assess structure–activity relationships for this new scaffold, a library of benzamide-containing novologues was prepared and evaluated against two breast cancer cell lines. Compound 14a manifested the most potent anti-proliferative activity from these studies and induced Hsp90-dependent client protein degradation in a concentration-dependent manner.

Synthesis and biological evaluation of coumarin replacements of novobiocin as Hsp90 inhibitors

Since Hsp90 modulates all six hallmarks of cancer simultaneously, it has become an attractive target for the development of cancer chemotherapeutics. In an effort to develop more efficacious compounds for Hsp90 inhibition, novobiocin analogues were prepared by replacing the central coumarin core with naphthalene, quinolinone, and quinoline surrogates. These modifications allowed for modification of the 2-position, which was previously unexplored. Biological evaluation of these compounds suggests a hydrophobic pocket about the 2-position of novobiocin. Anti-proliferative activities of these analogues against multiple cancer cell lines identified 2-alkoxyquinoline derivatives to exhibit improved activity.

Marine Sediment-Derived Streptomyces Bacteria from British Columbia, Canada Are a Promising Microbiota Resource for the Discovery of Antimicrobial Natural Products

Representatives of the genus Streptomyces from terrestrial sources have been the focus of intensive research for the last four decades because of their prolific production of chemically diverse and biologically important compounds. However, metabolite research from this ecological niche had declined significantly in the past years because of the rediscovery of the same bioactive compounds and redundancy of the sample strains. More recently, a new picture has begun to emerge in which marine-derived Streptomyces bacteria have become the latest hot spot as new source for unique and biologically active compounds. Here, we investigated the marine sediments collected in the temperate cold waters from British Columbia, Canada as a valuable source for new groups of marine-derived Streptomyces with antimicrobial activities. We performed culture dependent isolation from 49 marine sediments samples and obtained 186 Streptomyces isolates, 47 of which exhibited antimicrobial activities. Phylogenetic analyses of the active isolates resulted in the identification of four different clusters of bioactive Streptomyces including a cluster with isolates that appear to represent novel species. Moreover, we explored whether these marine-derived Streptomyces produce new secondary metabolites with antimicrobial properties. Chemical analyses revealed structurally diverse secondary metabolites, including four new antibacterial novobiocin analogues. We conducted structure-activity relationships (SAR) studies of these novobiocin analogues against methicillin-resistant Staphylococcus aureus (MRSA). In this study, we revealed the importance of carbamoyl and OMe moieties at positions 3” and 4” of novobiose as well as the hydrogen substituent at position 5 of hydroxybenzoate ring for the anti-MRSA activity. Changes in the substituents at these positions dramatically impede or completely eliminate the inhibitory activity of novobiocins against MRSA.

Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors

The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu.

Synthesis and biological evaluation of novobiocin analogues as potential heat shock protein 90 inhibitors

Recent studies have shown that novobiocin (NB), a member of the coumermycin (CA) family of antibiotics with demonstrated DNA gyrase inhibitory activity, inhibits Heat shock protein 90 (HSP90) by binding weakly to a putative ATP-binding site within its C-terminus. To develop more potent HSP90 inhibitors that target this site and to define structure–activity relationships (SARs) for this class of compounds, we have synthesized twenty seven 3-amido-7-noviosylcoumarin analogues starting from NB and CA. These were evaluated for evidence of HSP90 inhibition using several biological assays including inhibition of cell proliferation and cell cycle arrest, induction of the heat shock response, inhibition of luciferase-refolding in vitro, and depletion of the HSP90 client protein c-erbB-2/HER-2/neu (HER2). This SAR study revealed that a substantial increase in biological activity can be achieved by introduction of an indole-2-carboxamide group in place of 4-hydroxy-isopentylbenzamido group at C-3 of NB in addition to removal/derivatization of the 4-hydroxyl group from the coumarin ring. Methylation of the 4-hydroxyl group in the coumarin moiety moderately increased biological activity as shown by compounds 11 and 13. Our most potent new analogue 19 demonstrated biological activities consistent with known HSP90-binding agents, but with greater potency than NB.

3D-QSAR-Assisted Design, Synthesis, and Evaluation of Novobiocin Analogues

Hsp90 is an attractive therapeutic target for the treatment of cancer. Extensive structural modifications to novobiocin, the first Hsp90 C-terminal inhibitor discovered, have produced a library of novobiocin analogues and revealed some structure-activity relationships. Based upon the most potent novobiocin analogues generated from prior studies, a three-dimensional quantitative structure-activity (3D-QSAR) model was built. In addition, a new set of novobiocin analogues containing various structural features supported by the 3D-QSAR model were synthesized and evaluated against two breast cancer cell lines. Several new inhibitors produced anti-proliferative activity at mid nano-molar concentrations, which results through Hsp90 inhibition.

Novobiocin analogues with second-generation noviose surrogates

Hsp90 is a promising therapeutic target for the treatment of cancer. Novobiocin is the first Hsp90 C-terminal inhibitor ever identified and recent structure–activity relationship studies on the noviose sugar identified several commercially available amines as suitable surrogates. In an effort to further understand this region of the molecule, analogues containing various N′-amino substituents were prepared and evaluated against two breast cancer cell lines for determination of their efficacy. Compound 37j manifested the most potent anti-proliferative activity from these studies and induced Hsp90-dependent client protein degradation at mid nano-molar concentrations.