Compound 2 (KU-32) is a first-generation novologue (a novobiocin-based, C-terminal, heat shock protein 90 (Hsp90) inhibitor), that decreases glucose-induced death of primary sensory neurons and reverses numerous clinical indices of diabetic peripheral neuropathy in mice. The current study sought to exploit the C-terminal binding site of Hsp90 to determine whether the optimization of hydrogen bonding and hydrophobic interactions of second generation novologues could enhance neuroprotective activity. Using a series of substituted phenylboronic acids to replace the coumarin lactone of 2, we identified electronegative atoms placed at the meta-position of the B-ring exhibit improved cytoprotective activity, which is believed to result from favorable interactions with Lys539 in the Hsp90 C-terminal binding pocket. Consistent with these results, a meta-3-fluorophenyl substituted novologue (13b) exhibited a 14-fold lower ED50 compared to 2 for protection against glucose-induced toxicity of primary sensory neurons.
Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/β (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/β client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.
Heat shock protein 90 (Hsp90) has emerged as a promising therapeutic target for the treatment of cancer. Several Hsp90 inhibitors have entered clinical trials. However, some toxicological detriments have arisen, such as cardiotoxicity resulting from hERG inhibition following the administration of Hsp90 inhibitors. We sought to investigate this toxicity as hERG has been previously reported as a client protein that depends upon Hsp90 for its maturation and functional trafficking. In this study we show that hERG depends upon a single Hsp90 isoform. hERG preferentially co-immunoprecipitated with Hsp90α and genetic knockdown of Hsp90α, but not Hsp90β, resulted in a trafficking-defective hERG channel. This study demonstrates the importance of delineating the isoform dependence of Hsp90 client proteins and provides rationale for the design of isoform-selective Hsp90 inhibitors that avoid detrimental effects.
Hsp90; isoform; hERG
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.
Heat shock protein 90; Hsp90 inhibitors; Novobiocin; 3D-QSAR; Breast cancer
A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2±1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogs were synthesized from the abundant dimeric component of primuline. Some of the 29 analogs prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6±1 μM.
Hepatitis C virus; NS3; helicase; protease; ATPase; benzothiazoles
The potential therapeutic benefits associated with Hsp90 modulation for the treatment of cancer and neurodegenerative diseases highlight the importance of identifying novel Hsp90 scaffolds. KU-398, a novobiocin analogue, and silybin were recently identified as new Hsp90 inhibitors. Consequently, a library of 3-arylcoumarin derivatives that incorporated the structural features of KU-398 and silybin was designed, synthesized and evaluated against two breast cancer cell lines. Western blot analysis confirmed that the resulting 3-arylcoumarin hybrids target the Hsp90 protein folding machinery.
Heat shock protein 90; Hsp90 inhibitors; Novobiocin; Silybin; 3-Arylcoumarin; Breast cancer
Novobiocin analogs lacking labile glycosidic ether have been designed, synthesized and evaluated for Hsp90 inhibitory activity. Replacement of the synthetically complex noviose sugar with simple aromatic side chains produced analogs that maintain moderate cytotoxic activity against MCF7 and SkBR3 breast cancer cell-lines. Rationale for the preparation of des-noviose novobiocin analogs in addition to their synthesis and biological evaluation are presented herein.
KU-32 is a novel, novobiocin-based Hsp90 inhibitor that protects against neuronal glucotoxicity and reverses multiple clinical indices of diabetic peripheral neuropathy in a rodent model. However, any drug with potential for treating diabetic complications must also have no adverse effects on the function of pancreatic islets. Thus, the goal of the current study was to assess the effect of KU-32 on the in vitro viability and function of human islets. Treating human islets with KU-32 for 24 hours showed no toxicity as assessed using the alamarBlue assay. Confocal microscopy confirmed that with a minimum of 2-day exposure, KU-32 improved cellular viability by blocking apoptosis. Functionally, isolated human islets released more glucose-stimulated insulin when preincubated in KU-32. However, diabetic BKS-db/db mice, a model for type 2 diabetes, administered KU-32 for 10 weeks did not show any significant changes in blood glucose and insulin levels, despite having greater insulin staining/beta cell in the pancreas compared to untreated BKS db/db mice. In summary, KU-32 did not harm isolated human islets and may even be protective. However, the effect does not appear significant enough to alter the in vivo metabolic parameters of diabetic mice.
The half maximal inhibitory concentration (IC50) has several limitations that make it unsuitable for examining a large number of compounds in cytotoxicity studies, particularly when multiple exposure periods are tested. This article proposes a new approach to measure drug effectiveness, which allows ranking compounds according to their toxic effects on live cells. This effectiveness measure, which combines all exposure times tested, compares the growth rates of a particular cell line in the presence of the compound with its growth rate in the presence of DMSO alone. Our approach allows measuring a wider spectrum of toxicity than the IC50 approach, and allows automatic analyses of a large number of compounds. It can be easily implemented in linear regression software, provides a comparable measure of effectiveness for each investigated compound (both toxic and non-toxic), and allows statistically testing the null hypothesis that a compound is non-toxic versus the alternative that it is toxic. Importantly, our approach allows defining an automated decision rule for deciding whether a compound is significantly toxic. As an illustration, we describe the results of a cell-based study of the cytotoxicity of 24 analogs of novobiocin, a C-terminal inhibitor of heat shock protein 90 (Hsp90); the compounds were ranked in order of cytotoxicity to a panel of 18 cancer cell lines and 1 normal cell line. Our approach may also be a good alternative to computing the half maximal effective concentration (EC50) in studies searching for compounds that promote cell growth.
Anticancer drugs; cancer cell lines; cytotoxicity; drug effectiveness; EC50; exponential growth model; high throughput screening; hit selection; IC50
Macrocyclic natural products are a powerful class of lead-like chemical entities. Despite commonly violating Lipinski’s “rule of 5”, these compounds often demonstrate superior drug-like physicochemical and pharmacokinetic attributes when compared to their acyclic counterparts. However, the elaborate structural architectures of such molecules require rigorous synthetic investigation that complicates analogue development and their application to drug discovery programs. To circumvent these limitations, a conformation-based approach using limited SAR and molecular modeling was implemented to design simplified analogues of trienomycin A, in which the corresponding analogues could be prepared in a succinct manner to rapidly identify essential structural components necessary for biological activity. Trienomycin A is a member of the ansamycin family of natural products that possesses potent anticancer activity. These studies revealed a novel trienomycin A analogue, monoenomycin, which manifests potent anticancer activity.
Trienomycin A; Conformation; Anticancer; Ansamycin synthesis; Structure-activity relationship
The design, synthesis and biological evaluation of conformationally constrained coumermycin A1 analogues are reported. Compounds were evaluated against both breast cancer (SKBr3 and MCF7) and prostate cancer (PC3mm2, A549 and HT29) cell lines. Non-noviosylated coumermycin A1 analogues that manifest potent anti-proliferative activity resulting from Hsp90 inhibition are provided, wherein replacement of the stereochemically complex noviose sugar with readily available piperidine rings resulted in ~100 fold increase in anti-proliferative activities as compared to coumermycin A1, producing small molecule Hsp90 inhibitors that exhibit nanomolar activities.
Quantification of intra-epidermal nerve fibers (iENFs) is an important approach to stage diabetic peripheral neuropathy (DPN) and is a promising clinical endpoint for identifying beneficial therapeutics. Mechanistically, diabetes decreases neuronal mitochondrial function and enhancing mitochondrial respiratory capacity may aid neuronal recovery from glucotoxic insults. We have proposed that modulating the activity and expression of heat shock proteins (Hsp) may be of benefit in treating DPN. KU-32 is a C-terminal Hsp90 inhibitor that improved thermal hypoalgesia in diabetic C57Bl/6 mice but it was not determined if this was associated with an increase in iENF density and mitochondrial function. After 16 weeks of diabetes, Swiss Webster mice showed decreased electrophysiological and psychosensory responses and a >30% loss of iENFs. Treatment of the mice with ten weekly doses of 20 mg/kg KU-32 significantly reversed pre-existing deficits in nerve conduction velocity and responses to mechanical and thermal stimuli. KU-32 therapy significantly reversed the pre-existing loss of iENFs despite the identification of a sub-group of drug-treated diabetic mice that showed improved thermal sensitivity but no increase in iENF density. To determine if the improved clinical indices correlated with enhanced mitochondrial activity, sensory neurons were isolated and mitochondrial bioenergetics assessed ex vivo using extracellular flux technology. Diabetes decreased maximal respiratory capacity in sensory neurons and this deficit was improved following KU-32 treatment. In conclusion, KU-32 improved physiological and morphologic markers of degenerative neuropathy and drug efficacy may be related to enhanced mitochondrial bioenergetics in sensory neurons.
Intra-epidermal nerve fibers; Sensory neurons; Nerve conduction velocity; Mitochondria; Bioenergetics; Respiratory capacity
The Hsp90 chaperone machine is required for the folding, activation and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains, however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein.
Diabetic peripheral neuropathy (DPN) is a common complication of diabetes in which hyperglycemia-induced mitochondrial dysfunction and enhanced oxidative stress contribute to sensory neuron pathology. KU-32 is a novobiocin-based, C-terminal inhibitor of the molecular chaperone, heat shock protein 90 (Hsp90). KU-32 ameliorates multiple sensory deficits associated with the progression of DPN and protects unmyelinated sensory neurons from glucose-induced toxicity. Mechanistically, KU-32 increased the expression of Hsp70 and this protein was critical for drug efficacy in reversing DPN. However, it remained unclear if KU-32 had a broader effect on chaperone induction and if its efficacy was linked to improving mitochondrial dysfunction. Using cultures of hyperglycemically stressed primary sensory neurons, the present study investigated whether KU-32 had an effect on the translational induction of other chaperones and improved mitochondrial oxidative stress and bioenergetics. A variation of stable isotope labeling with amino acids in cell culture called pulse SILAC (pSILAC) was used to unbiasedly assess changes in protein translation. Hyperglycemia decreased the translation of numerous mitochondrial proteins that affect superoxide levels and respiratory activity. Importantly, this correlated with a decrease in mitochondrial oxygen consumption and an increase in superoxide levels. KU-32 increased the translation of Mn superoxide dismutase and several cytosolic and mitochondrial chaperones. Consistent with these changes, KU-32 decreased mitochondrial superoxide levels and significantly enhanced respiratory activity. These data indicate that efficacy of modulating molecular chaperones in DPN may be due in part to improved neuronal mitochondrial bioenergetics and decreased oxidative stress.
diabetes; hyperglycemia; SILAC; chaperones; sensory neurons; mitochondria; superoxide dismutase; oxidative stress
Development of the DNA gyrase inhibitor, novobiocin, into a selective Hsp90 inhibitor was accomplished through structural modifications to the amide side chain, coumarin ring, and sugar moiety. These species exhibit ~700-fold improved anti-proliferative activity versus the natural product as evaluated by cellular efficacies against breast, colon, prostate, lung, and other cancer cell lines. Utilization of structure–activity relationships established for three novobiocin synthons produced optimized scaffolds, which manifest mid-nanomolar activity against a panel of cancer cell lines and serve as lead compounds that manifest their activities through Hsp90 inhibition.
A high-throughput screening of natural product libraries identified (−)-gambogic acid (1), a component of the exudate of Garcinia harburyi, as a potential Hsp90 inhibitor, in addition to the known Hsp90 inhibitor celastrol (2). Subsequent testing established that 1 inhibited cell proliferation, brought about the degradation of Hsp90 client proteins in cultured cells, and induced the expression of Hsp70 and Hsp90, which are hallmarks of Hsp90 inhibition. Gambogic acid also disrupted the interaction of Hsp90, Hsp70 and Cdc37 with the heme-regulated eIF2α kinase (HRI- an Hsp90-dependent client) and blocked the maturation of HRI in vitro. Surface plasmon resonance spectroscopy indicated that 1 bound to the N-terminal domain of Hsp90 with a low micromolar Kd, in a manner that was not competitive with the Hsp90 inhibitor geldanamycin (3). Molecular docking experiments supported the posit that 1 binds Hsp90 at a site distinct from Hsp90’s ATP binding pocket. The data obtained have firmly established 1 as a novel Hsp90 inhibitor and have provided evidence of a new site that can be targeted for the development of improved Hsp90 inhibitors.
Dysfunctional insulin and insulin-like growth factor-I (IGF-I) signaling contributes to the pathological progression of diabetes, diabetic peripheral neuropathy (DPN), Alzheimer's (AD), Parkinson's (PD) and Huntington's diseases (HD). Despite their prevalence, there are limited therapeutic options available for the treatment of these neurodegenerative disorders. Therefore, establishing a link between insulin/IGF-I and the pathoetiology of these diseases may provide alternative approaches toward their management. Many of the heat shock proteins (Hsps) are well-known molecular chaperones that solubilize and clear damaged proteins and protein aggregates. Recent studies suggest that modulating Hsps may represent a promising therapeutic avenue for improving insulin and IGF-I signaling. Pharmacological induction of the heat shock response (HSR) may intersect with insulin/IGF-I signaling to improve aspects of neurodegenerative phenotypes. Herein, we review the intersection between Hsps and the insulin/IGF systems under normal and pathological conditions. The discussion will emphasize the potential of non-toxic HSR inducers as viable therapeutic agents.
Bacterial histidine kinases (HK) are members of the GHKL superfamily, which share a unique adenosine triphosphate (ATP)-binding Bergerat fold. Our previous studies have shown that Gyrase, Hsp90, MutL (GHL) inhibitors bind to the ATP-binding pocket of HK and may provide lead compounds for the design of novel antibiotics targeting these kinases. In this article, we developed a competition assay using the fluorescent ATP analog, 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate. The method can be used for high-throughput screening of compound libraries targeting HKs or other ATP-binding proteins. We utilized the assay to screen a library of GHL inhibitors targeting the bacterial HK PhoQ, and discuss the applications of the 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate competition assay beyond GHKL inhibitor screening.
Modulating molecular chaperones is emerging as an attractive approach to treat neurodegenerative diseases associated with protein aggregation, DPN (diabetic peripheral neuropathy) and possibly, demyelinating neuropathies. KU-32 [N-(7-((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy)-8-methyl-2-oxo-2H-chromen-3-yl)acetamide] is a small molecule inhibitor of Hsp90 (heat shock protein 90) and reverses sensory deficits associated with myelinated fibre dysfunction in DPN. Additionally, KU-32 prevented the loss of myelinated internodes induced by treating myelinated SC (Schwann cell)-DRG (dorsal root ganglia) sensory neuron co-cultures with NRG1 (neuregulin-1 Type 1). Since KU-32 decreased NRG1-induced demyelination in an Hsp70-dependent manner, the goal of the current study was to clarify how Hsp70 may be mechanistically linked to preventing demyelination. The activation of p42/p44 MAPK (mitogen-activated protein kinase) and induction of the transcription factor c-Jun serve as negative regulators of myelination. NRG1 activated MAPK, induced c-Jun expression and promoted a loss of myelin segments in DRG explants isolated from both WT (wild-type) and Hsp70 KO (knockout) mice. Although KU-32 did not block the activation of MAPK, it blocked c-Jun induction and protected against a loss of myelinated segments in WT mice. In contrast, KU-32 did not prevent the NRG1-dependent induction of c-Jun and loss of myelin segments in explants from Hsp70 KO mice. Overexpression of Hsp70 in myelinated DRG explants prepared from WT or Hsp70 KO mice was sufficient to block the induction of c-Jun and the loss of myelin segments induced by NRG1. Lastly, inhibiting the proteasome prevented KU-32 from decreasing c-Jun levels. Collectively, these data support that Hsp70 induction is sufficient to prevent NRG1-induced demyelination by enhancing the proteasomal degradation of c-Jun.
diabetic neuropathy; molecular chaperones; myelin basic protein; Schwann cells; sensory neurons; CHIP, C-terminus Hsp70-interacting protein; CMT1, Charcot–Marie–Tooth disease type 1; DAPI, 4,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; DPN, diabetic peripheral neuropathy; DRG, dorsal root ganglia; FCS, fetal calf serum; HRP, horseradish peroxidise; HS, heat shock; Hsc70, heat-shock cognate 70 stress protein; HSF1, heat shock factor 1; Hsp, heat shock protein; HSR, heat shock response; JNK, c-Jun N-terminal kinase; KO, knockout; KU-32, N-(7-((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy)-8-methyl-2-oxo-2H-chromen-3-yl)acetamide; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; NRG1, neuregulin-1 Type 1; pAb, polyclonal antibody; PBST, phosphate-buffered saline containing 0.1% Tween 20; PGP9.5, protein gene product 9.5; phospho-c-Jun, phosphorylated c-Jun; PMP22, peripheral myelin protein 22; SC, Schwann cell; WT, wild-type
Structural modifications to the coumarin core and benzamide side chain of novobiocin have successfully transformed the natural product from a selective DNA gyrase inhibitor into a potent inhibitor of the Hsp90 C-terminus. However, no SAR studies have been conducted on the noviose appendage, which represents the rate-limiting synthon in the preparation of analogues. Therefore, a series of sugar mimics and non-sugar derivatives were synthesized and evaluated to identify simplified compounds that exhibit Hsp90 inhibition. Evaluation against two breast cancer cell lines demonstrated that replacement of the stereochemical complex noviose with simplified alkyl amines increased anti-proliferative activity, resulting in novobiocin analogues that manifest IC50 values in the mid nanomolar range.
Heat shock protein 90; Hsp90 inhibitors; Novobiocin; Stucture-activity relationships; Breast cancer
There is much recent interest in the application of copper-free click chemistry to study a wide range of biological events in vivo and in vitro. Specifically, azide-conjugated fluorescent probes can be used to identify targets which have been modified with bioorthogonal reactive groups. For intracellular applications of this chemistry, the structural and physicochemical properties of the fluorescent azide become increasingly important. Ideal fluorophores should extensively accumulate within cells, have even intracellular distribution, and be free (unbound), allowing them to efficiently participate in bimolecular reactions. We report here on the synthesis and evaluation a set of structurally diverse fluorescent probes to examine their potential usefulness in intracellular click reactions. Total cellular uptake and intracellular distribution profiles were comparatively assessed using both quantitative and qualitative approaches. The intracellular diffusion coefficients were measured using a fluorescence recovery after photobleaching (FRAP)-based method. Many reactive fluorophores exhibited suboptimal properties for intracellular reactions. BODIPY- and TAMRA-based azides had superior cellular accumulation, whereas TAMRA-based probes had the most uniform intracellular distribution and best cytosolic diffusivity. Collectively, these results provide an unbiased comparative evaluation regarding the suitability of azide-linked fluorophores for intracellular click reactions.
Fluorescent dyes; tetramethylrhodamine; BODIPY; click chemistry; FRAP analysis; fluorescence microscopy
A series of triazole-containing novobiocin analogues has been designed, synthesized and their inhibitory activity determined. These compounds contain a triazole ring in lieu of the amide moiety present in the natural product. The anti-proliferative effects of these compounds were evaluated against two breast cancer cell lines (SKBr-3 and MCF-7), and manifested activities similar to their amide-containing counterparts. In addition, Hsp90-dependent client protein degradation was observed via western blot analysis, further supporting a common mode of Hsp90 inhibition for both structural classes.
Hsp90; Novobiocin; Antiproliferation; Triazole
The natural products novobiocin and derrubone have both demonstrated Hsp90 inhibition and structure–activity relationships have been established for each scaffold. Given these compounds share several key structural features, we hypothesized that incorporation of elements from each could provide insight to structural features important for Hsp90 inhibition. Thus, chimeric analogues of novobiocin and derrubone were constructed and evaluated. These studies confirmed that the functionality present at the 3-position of the isoflavone plays a critical role in determining Hsp90 inhibition and suggests that the bicyclic ring system present in both novobiocin and derrubone do not share similar modes of binding.
Hsp90; novobiocin; derrubone; anticancer
Bicyclic radester analogues have been synthesized and evaluated for Hsp90 inhibitory activity. These analogues induce concentration-dependent degradation of Hsp90-dependent client proteins with the six-membered bicyclic analogues manifesting increased activity versus the five-membered counterparts.
Hsp90; Geldanamycin; Radicicol; Anti-cancer