Hepatocyte-targeted siTAZ therapy lowers liver fibrosis in NASH diet-fed chimeric mice with hepatocyte-humanized livers.

Nonalcoholic steatohepatitis (NASH) is emerging as the most common cause of liver disease. Although many studies in mouse NASH models have suggested therapies, translation to humans is poor, with no approved drugs for NASH. One explanation may lie in differences between mouse and human hepatocytes. We used NASH diet-fed chimeric mice reconstituted with human hepatocytes (hu-liver mice) to test a mechanism-based hepatocyte-targeted small interfering RNA (siRNA), GalNAc-siTaz, shown previously to block the progression to fibrotic NASH in mice. Following ablation of endogenous hepatocytes, male mice were reconstituted with human hepatocytes from a single donor with the rs738409-C/G PNPLA3 risk variant, resulting in ∼95% human hepatocyte reconstitution. The mice were then fed a high-fat choline-deficient l-amino acid-defined diet for 6 weeks to induce NASH, followed by six weekly injections of GalNAc-siTAZ to silence hepatocyte-TAZ or control GalNAc-siRNA (GalNAc-control) while still on the NASH diet. GalNAc-siTAZ lowered human hepatic TAZ and IHH, a TAZ target that promotes NASH fibrosis. Most important, GalNAc-siTAZ decreased liver inflammation, hepatocellular injury, hepatic fibrosis, and profibrogenic mediator expression versus GalNAc-control, indicating that GalNAc-siTAZ decreased the progression of NASH in mice reconstituted with human hepatocytes. In conclusion, silencing TAZ in human hepatocytes suppresses liver fibrosis in a hu-liver model of NASH.

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.

Simultaneous measurement of mouse and human albumin in chimeric mice with humanized livers.

Background: The degree of human hepatocyte replacement in chimeric mice with humanized liver has previously been shown to correlate with human plasma albumin measurements. However, there are no reports that directly compare the remaining endogenous mouse albumin with the newly expressed human albumin following engraftment. To better understand the disposition of serum albumin in PXB-mice, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to simultaneously quantitate both human and mouse albumin from plasma. Results: A robust correlation was observed between the serum human albumin levels measured by LC-MS/MS and the estimated replacement index of PXB-mice. Conclusion: All data were shown to be specific and suitable to accurately quantify both human and mouse albumin from plasma of chimeric mice with humanized livers.

Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model.

BACKGROUND & AIMS: Infection with hepatitis C virus (HCV) remains a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, there is no approved therapeutic targeting this potentially druggable biomarker. METHODS: The anti-HCV activity of a fusion protein consisting of Avaren lectin and the fragment crystallizable (Fc) region of a human immunoglobulin G1 antibody, Avaren-Fc (AvFc) was evaluated through the use of in vitro neutralization assays as well as an in vivo challenge in a chimeric human liver (PXB) mouse model. Drug toxicity was assessed by histopathology, serum alanine aminotransferase, and mouse body weights. RESULTS: AvFc was capable of neutralizing cell culture-derived HCV in a genotype-independent manner, with 50% inhibitory concentration values in the low nanomolar range. Systemic administration of AvFc in a histidine-based buffer was well tolerated; after 11 doses every other day at 25 mg/kg there were no significant changes in body or liver weights or in blood human albumin or serum alanine aminotransferase activity. Gross necropsy and liver pathology confirmed the lack of toxicity. This regimen successfully prevented genotype 1a HCV infection in all animals, although an AvFc mutant lacking HMG binding activity failed. CONCLUSIONS: These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection, and AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

Prediction of Human Disproportionate and Biliary Excreted Metabolites Using Chimeric Mice with Humanized Liver.

The PXB-mouse is potentially a useful in vivo model to predict human hepatic metabolism and clearance. Four model compounds, [14C]desloratadine, [3H]mianserin, cyproheptadine, and [3H]carbazeran, all reported with disproportionate human metabolites, were orally administered to PXB- or control SCID mice to elucidate the biotransformation of each of them. For [14C]desloratadine in PXB-mice, O-glucuronide of 3-hydroxydesloratadine was observed as the predominant metabolite in both the plasma and urine. Both 3-hydroxydesloratadine and its O-glucuronide were detected as major drug-related materials in the bile, whereas only 3-hydroxydesloratadine was detected in the feces, suggesting that a fraction of 3-hydroxydesloratadine in feces was derived from deconjugation of its O-glucuronide by gut microflora. This information can help understand the biliary clearance mechanism of a drug and may fill the gap in a human absorption, distribution, metabolism, and excretion study, in which the bile samples are typically not available. The metabolic profiles in PXB-mice were qualitatively similar to those reported in humans in a clinical study in which 3-hydroxydesloratadine and its O-glucuronide were major and disproportionate metabolites compared with rat, mouse, and monkey. In the control SCID mice, neither of the metabolites was detected in any matrix. Similarly, for the other three compounds, all human specific or disproportionate metabolites were detected at a high level in PXB-mice, but they were either minimally observed or not observed in the control mice. Data from these four compounds indicate that studies in PXB-mice can help predict the potential for the presence of human disproportionate metabolites (relative to preclinical species) prior to conducting clinical studies and understand the biliary clearance mechanism of a drug. SIGNIFICANCE STATEMENT: Studies in PXB-mice have successfully predicted the human major and disproportionate metabolites compared with preclinical safety species for desloratadine, mianserin, cyproheptadine, and carbazeran. In addition, biliary excretion data from PXB-mice can help illustrate the human biliary clearance mechanism of a drug.

Design, Synthesis, and Biological Evaluation of 4-Quinoline Carboxylic Acids as Inhibitors of Dihydroorotate Dehydrogenase.

We pursued a structure-guided approach toward the development of improved dihydroorotate dehydrogenase (DHODH) inhibitors with the goal of forming new interactions between DHODH and the brequinar class of inhibitors. Two potential residues, T63 and Y356, suitable for novel H-bonding interactions, were identified in the brequinar-binding pocket. Analogues were designed to maintain the essential pharmacophore and form new electrostatic interactions through strategically positioned H-bond accepting groups. This effort led to the discovery of potent quinoline-based analogues 41 (DHODH IC50 = 9.71 ± 1.4 nM) and 43 (DHODH IC50 = 26.2 ± 1.8 nM). A cocrystal structure between 43 and DHODH depicts a novel water mediated H-bond interaction with T63. Additional optimization led to the 1,7-naphthyridine 46 (DHODH IC50 = 28.3 ± 3.3 nM) that forms a novel H-bond with Y356. Importantly, compound 41 possesses significant oral bioavailability ( F = 56%) and an elimination t1/2 = 2.78 h (PO dosing). In conclusion, the data supports further preclinical studies of our lead compounds toward selection of a candidate for early-stage clinical development.

Intradiscal injection of simvastatin results in radiologic, histologic, and genetic evidence of disc regeneration in a rat model of degenerative disc disease.

BACKGROUND CONTEXT: A large percentage of back pain can be attributed to degeneration of the intervertebral disc (IVD). Bone morphogenetic protein 2 (BMP-2) is known to play an important role in chondrogenesis of the IVD. Simvastatin is known to upregulate expression of BMP-2. Thus, we hypothesized that intradiscal injection of simvastatin in a rat model of degenerative disc disease (DDD) would result in retardation of DDD. PURPOSE: The purpose of the present study was to develop a novel conservative treatment for DDD and related discogenic back pain. STUDY DESIGN/SETTING: The setting of this study is the laboratory investigation. METHODS: Disc injury was induced in 272 rats via 21-ga needle puncture. After 6 weeks, injured discs were treated with simvastatin in a saline or hydrogel carrier. Rats were sacrificed at predetermined time points. Outcome measures assessed were radiologic, histologic, and genetic. Radiologically, the magnetic resonance imaging (MRI) index (number of pixels multiplied by the corresponding image densities) was determined. Histologically, disc spaces were read by three blinded scorers using a previously described histologic grading scale. Genetically, nuclei pulposi were harvested, and polymerase chain reaction was run to determine relative levels of aggrecan, collagen type II, and BMP-2 gene expression. RESULTS: Radiologically, discs treated with 5 mg/mL of simvastatin in hydrogel or saline demonstrated MRI indices that were normal through 8 weeks after treatment, although this was more sustained when delivered in hydrogel. Histologically, discs treated with 5 mg/mL of simvastatin in hydrogel demonstrated improved grades compared with discs treated at higher doses. Genetically, discs treated with 5 mg/mL of simvastatin in hydrogel demonstrated higher gene expression of aggrecan and collagen type II than control. CONCLUSIONS: Degenerate discs treated with 5 mg/mL of simvastatin in a hydrogel carrier demonstrated radiographic and histologic features resembling normal noninjured IVDs. In addition, the gene expression of aggrecan and collagen type II (important constituents of the IVD extracellular matrix) was upregulated in treated discs. Injection of simvastatin into degenerate IVDs may result in retardation of disc degeneration and represents a promising investigational therapy for conservative treatment of DDD.

Allosteric heat shock protein 70 inhibitors rapidly rescue synaptic plasticity deficits by reducing aberrant tau.

BACKGROUND: The microtubule-associated protein tau accumulates in neurodegenerative diseases known as tauopathies, the most common being Alzheimer's disease. One way to treat these disorders may be to reduce abnormal tau levels through chaperone manipulation, thus subverting synaptic plasticity defects caused by tau's toxic accretion. METHODS: Tauopathy models were used to study the impact of YM-01 on tau. YM-01 is an allosteric promoter of triage functions of the most abundant variant of the heat shock protein 70 (Hsp70) family in the brain, heat shock cognate 70 protein (Hsc70). The mechanisms by which YM-01 modified Hsc70 activity and tau stability were evaluated with biochemical methods, cell cultures, and primary neuronal cultures from tau transgenic mice. YM-01 was also administered to acute brain slices of tau mice; changes in tau stability and electrophysiological correlates of learning and memory were measured. RESULTS: Tau levels were rapidly and potently reduced in vitro and ex vivo upon treatment with nanomolar concentrations of YM-01. Consistent with Hsc70 having a key role in this process, overexpression of heat shock protein 40 (DNAJB2), an Hsp70 co-chaperone, suppressed YM-01 activity. In contrast to its effects in pathogenic tauopathy models, YM-01 had little activity in ex vivo brain slices from normal, wild-type mice unless microtubules were disrupted, suggesting that Hsc70 acts preferentially on abnormal pools of free tau. Finally, treatment with YM-01 increased long-term potentiation in tau transgenic brain slices. CONCLUSIONS: Therapeutics that exploit the ability of chaperones to selectively target abnormal tau can rapidly and potently rescue the synaptic dysfunction that occurs in Alzheimer's disease and other tauopathies.

Synthesis and initial evaluation of YM-08, a blood-brain barrier permeable derivative of the heat shock protein 70 (Hsp70) inhibitor MKT-077, which reduces tau levels.

The molecular chaperone, heat shock protein 70 (Hsp70), is an emerging drug target for treating neurodegenerative tauopathies. We recently found that one promising Hsp70 inhibitor, MKT-077, reduces tau levels in cellular models. However, MKT-077 does not penetrate the blood-brain barrier (BBB), limiting its use as either a clinical candidate or probe for exploring Hsp70 as a drug target in the central nervous system (CNS). We hypothesized that replacing the cationic pyridinium moiety in MKT-077 with a neutral pyridine might improve its clogP and enhance its BBB penetrance. To test this idea, we designed and synthesized YM-08, a neutral analogue of MKT-077. Like the parent compound, YM-08 bound to Hsp70 in vitro and reduced phosphorylated tau levels in cultured brain slices. Pharmacokinetic evaluation in CD1 mice showed that YM-08 crossed the BBB and maintained a brain/plasma (B/P) value of ∼0.25 for at least 18 h. Together, these studies suggest that YM-08 is a promising scaffold for the development of Hsp70 inhibitors suitable for use in the CNS.

Activation of Hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation.

We sought new strategies to reduce amounts of the polyglutamine androgen receptor (polyQ AR) and achieve benefits in models of spinobulbar muscular atrophy, a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the heat shock protein 90 (Hsp90)- and Hsp70-based chaperone machinery, but mechanisms regulating the protein's turnover are incompletely understood. We demonstrate that overexpression of Hsp70 interacting protein (Hip), a co-chaperone that enhances binding of Hsp70 to its substrates, promotes client protein ubiquitination and polyQ AR clearance. Furthermore, we identify a small molecule that acts similarly to Hip by allosterically promoting Hsp70 binding to unfolded substrates. Like Hip, this synthetic co-chaperone enhances client protein ubiquitination and polyQ AR degradation. Both genetic and pharmacologic approaches targeting Hsp70 alleviate toxicity in a Drosophila model of spinobulbar muscular atrophy. These findings highlight the therapeutic potential of allosteric regulators of Hsp70 and provide new insights into the role of the chaperone machinery in protein quality control.

Cysteine reactivity distinguishes redox sensing by the heat-inducible and constitutive forms of heat shock protein 70.

The heat shock protein 70 (Hsp70) family of molecular chaperones has important functions in maintaining proteostasis under stress conditions. Several Hsp70 isoforms, especially Hsp72 (HSPA1A), are dramatically upregulated in response to stress; however, it is unclear whether these family members have biochemical properties that are specifically adapted to these scenarios. The redox-active compound, methylene blue (MB), has been shown to inhibit the ATPase activity of Hsp72 in vitro, and it promotes degradation of the Hsp72 substrate, tau, in cellular and animal models. Here, we report that MB irreversibly inactivates Hsp72 but not the nearly identical, constitutively expressed isoform, heat shock cognate 70 (Hsc70; HSPA8). Mass spectrometry results show that MB oxidizes Cys306, which is not conserved in Hsc70. Molecular models suggested that oxidation of Cys306 exposes Cys267 to modification and that both events contribute to loss of ATP binding in response to MB. Consistent with this model, mutating Cys267 and Cys306 to serine made Hsp72 largely resistant to MB in vitro, and overexpression of the C306S mutant blocked MB-mediated loss of tau in a cellular model. Furthermore, mutating Cys267 and Cys306 to the pseudo-oxidation mimic, aspartic acid, mirrored MB treatment: the C267D and C306D mutants had reduced ATPase activity in vitro, and overexpression of the C267/306D double mutant significantly reduced tau levels in cells. Together, these results suggest that redox sensing by specific cysteine residues in Hsp72, but not Hsc70, may be an important component of the chaperone response to oxidative stress.

A screen for modulators of large T antigen's ATPase activity uncovers novel inhibitors of Simian Virus 40 and BK virus replication.

New polyomaviruses are continually being identified, and it is likely that links between this virus family and disease will continue to emerge. Unfortunately, a specific treatment for polyomavirus-associated disease is lacking. Because polyomaviruses express large Tumor Antigen, TAg, we hypothesized that small molecule inhibitors of the essential ATPase activity of TAg would inhibit viral replication. Using a new screening platform, we identified inhibitors of TAg's ATPase activity. Lead compounds were moved into a secondary assay, and ultimately two FDA approved compounds, bithionol and hexachlorophene, were identified as the most potent TAg inhibitors known to date. Both compounds inhibited Simian Virus 40 replication as assessed by plaque assay and quantitative PCR. Moreover, these compounds inhibited BK virus, which causes BKV Associated Nephropathy. In neither case was host cell viability compromised at these concentrations. Our data indicate that directed screening for TAg inhibitors is a viable method to identify polyomavirus inhibitors, and that bithionol and hexachlorophene represent lead compounds that may be further modified and/or ultimately used to combat diseases associated with polyomavirus infection.

Rhodacyanine derivative selectively targets cancer cells and overcomes tamoxifen resistance.

MKT-077, a rhodacyanine dye, was shown to produce cancer specific cell death. However, complications prevented the use of this compound beyond clinical trials. Here we describe YM-1, a derivative of MKT-077. We found that YM-1 was more cytotoxic and localized differently than MKT-077. YM-1 demonstrated this cytotoxicity across multiple cancer cell lines. This toxicity was limited to cancer cell lines; immortalized cell models were unaffected. Brief applications of YM-1 were found to be non-toxic. Brief treatment with YM-1 restored tamoxifen sensitivity to a refractory tamoxifen-resistant MCF7 cell model. This effect is potentially due to altered estrogen receptor alpha phosphorylation, an outcome precipitated by selective reductions in Akt levels (Akt/PKB). Thus, modifications to the rhodocyanine scaffold could potentially be made to improve efficacy and pharmacokinetic properties. Moreover, the impact on tamoxifen sensitivity could be a new utility for this compound family.

Molecular chaperones and regulation of tau quality control: strategies for drug discovery in tauopathies.

Tau is a microtubule-associated protein that accumulates in at least 15 different neurodegenerative disorders, which are collectively referred to as tauopathies. In these diseases, tau is often hyperphosphorylated and found in aggregates, including paired helical filaments, neurofibrillary tangles and other abnormal oligomers. Tau aggregates are associated with neuron loss and cognitive decline, which suggests that this protein can somehow evade normal quality control allowing it to aberrantly accumulate and become proteotoxic. Consistent with this idea, recent studies have shown that molecular chaperones, such as heat shock protein 70 and heat shock protein 90, counteract tau accumulation and neurodegeneration in disease models. These molecular chaperones are major components of the protein quality control systems and they are specifically involved in the decision to retain or degrade many proteins, including tau and its modified variants. Thus, one potential way to treat tauopathies might be to either accelerate interactions of abnormal tau with these quality control factors or tip the balance of triage towards tau degradation. In this review, we summarize recent findings and suggest models for therapeutic intervention.

Palmerolide macrolides from the Antarctic tunicate Synoicum adareanum.

Palmerolides D-G are new bioactive macrolides isolated from the Antarctic tunicate Synoicum adareanum and are related to the melanoma-selective cytotoxin palmerolide A. Most of these palmerolides are potent V-ATPase inhibitors and have sub-micromolar activity against melanoma. Though palmerolide A remains the most potent of this series of natural products against mammalian V-ATPase, recent data suggests that palmerolide D is the most potent against melanoma. A comparison of the bioactivity data obtained for these natural product palmerolides has provided insight into the substructures necessary to retain V-ATPase inhibition and cytotoxic activity.

Heme-dependent activation of neuronal nitric oxide synthase by cytosol is due to an Hsp70-dependent, thioredoxin-mediated thiol-disulfide interchange in the heme/substrate binding cleft.

We have reported that heme-dependent activation of apo-neuronal nitric oxide synthase (apo-nNOS) to the active holo-enzyme dimer is dependent upon factors present in reticulocyte lysate and other cytosols. Here, we find that both Hsp70 and thioredoxin are components of the activation system. The apo-nNOS activating activity of reticulocyte lysate is retained in a pool of fractions containing Hsp70 that elute from DE52 prior to Hsp90. All of the activating activity and 20-30% of the Hsp70 elute in the flow-through fraction upon subsequent ATP-agarose chromatography. Apo-nNOS activation by this flow-through fraction is inhibited by pifithrin-µ, a small molecule inhibitor of Hsp70, suggesting that a non-ATP-binding form of Hsp70 is involved in heme-dependent apo-nNOS activation. Previous work has shown that apo-nNOS can be activated by thiol-disulfide exchange, and we show substantial activation with a small molecule dithiol modeled on the active motifs of thioredoxin and protein disulfide isomerase. Further fractionation of the ATP-agarose flow-through on Sephacryl S-300 separates free thioredoxin from apo-nNOS activating activity, Hsp70, and a small amount of thioredoxin, all of which are eluted throughout the macromolecular peak. Incubation of apo-nNOS with the macromolecular fraction in combination either with the thioredoxin-containing fraction or with purified recombinant human thioredoxin restores full heme-dependent activating activity. This supports a model in which Hsp70 binding to apo-nNOS stabilizes an open state of the heme/substrate binding cleft to facilitate thioredoxin access to the active site cysteine that coordinates with heme iron, permitting heme binding and dimerization to the active enzyme.

Allosteric drugs: the interaction of antitumor compound MKT-077 with human Hsp70 chaperones.

Hsp70 (heat shock protein 70 kDa) chaperones are key to cellular protein homeostasis. However, they also have the ability to inhibit tumor apoptosis and contribute to aberrant accumulation of hyperphosphorylated tau in neuronal cells affected by tauopathies, including Alzheimer's disease. Hence, Hsp70 chaperones are increasingly becoming identified as targets for therapeutic intervention in these widely abundant diseases. Hsp70 proteins are allosteric machines and offer, besides classical active-site targets, also opportunities to target the mechanism of allostery. In this work, it is demonstrated that the action of the potent anticancer compound MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride) occurs through a differential interaction with Hsp70 allosteric states. MKT-077 is therefore an "allosteric drug." Using NMR spectroscopy, we identify the compound's binding site on human HSPA8 (Hsc70). The binding pose is obtained from NMR-restrained docking calculations, subsequently scored by molecular-dynamics-based energy and solvation computations. Suggestions for the improvement of the compound's properties are made on the basis of the binding location and pose.

Chemical screens against a reconstituted multiprotein complex: myricetin blocks DnaJ regulation of DnaK through an allosteric mechanism.

DnaK is a molecular chaperone responsible for multiple aspects of bacterial proteostasis. The intrinsically slow ATPase activity of DnaK is stimulated by its co-chaperone, DnaJ, and these proteins often work in concert. To identify inhibitors we screened plant-derived extracts against a reconstituted mixture of DnaK and DnaJ. This approach resulted in the identification of flavonoids, including myricetin, which inhibited activity by up to 75%. Interestingly, myricetin prevented DnaJ-mediated stimulation of ATPase activity, with minimal impact on either DnaK's intrinsic turnover rate or its stimulation by another co-chaperone, GrpE. Using NMR, we found that myricetin binds DnaK at an unanticipated site between the IB and IIB subdomains and that it allosterically blocked binding of DnaK to DnaJ. Together, these results highlight a "gray box" screening approach, which might facilitate the identification of inhibitors of other protein-protein interactions.

High-throughput screen for Escherichia coli heat shock protein 70 (Hsp70/DnaK): ATPase assay in low volume by exploiting energy transfer.

Members of the heat shock protein 70 (Hsp70) family of molecular chaperones are emerging as potential therapeutic targets. Their ATPase activity has classically been measured using colorimetric phosphate detection reagents, such as quinaldine red (QR). Although such assays are suitable for 96-well plate formats, they typically lose sensitivity when attempted in lower volume due to path length and meniscus effects. These limitations and Hsp70's weak enzymatic activity have combined to create significant challenges in high-throughput screening. To overcome these difficulties, the authors have adopted an energy transfer strategy that was originally reported by Zuck et al. (Anal Biochem 2005;342:254-259). Briefly, white 384-well plates emit fluorescence when irradiated at 430 nm. In turn, this intrinsic fluorescence can be quenched by energy transfer with the QR-based chromophore. Using this more sensitive approach, the authors tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z' ~0.6, coefficient of variation ~8%), and at least one promising new inhibitor was identified. In secondary assays, this compound specifically blocked stimulation of DnaK by its co-chaperone, DnaJ. Thus, this simple and inexpensive adaptation of a colorimetric method might be suitable for screening against Hsp70 family members.

Hsc70 rapidly engages tau after microtubule destabilization.

The microtubule-associated protein Tau plays a crucial role in regulating the dynamic stability of microtubules during neuronal development and synaptic transmission. In a group of neurodegenerative diseases, such as Alzheimer disease and other tauopathies, conformational changes in Tau are associated with the initial stages of disease pathology. Folding of Tau into the MC1 conformation, where the amino acids at residues 7-9 interact with residues 312-342, is one of the earliest pathological alterations of Tau in Alzheimer disease. The mechanism of this conformational change in Tau and the subsequent effect on function and association to microtubules is largely unknown. Recent work by our group and others suggests that members of the Hsp70 family play a significant role in Tau regulation. Our new findings suggest that heat shock cognate (Hsc) 70 facilitates Tau-mediated microtubule polymerization. The association of Hsc70 with Tau was rapidly enhanced following treatment with microtubule-destabilizing agents. The fate of Tau released from the microtubule was found to be dependent on ATPase activity of Hsc70. Microtubule destabilization also rapidly increased the MC1 folded conformation of Tau. An in vitro assay suggests that Hsc70 facilitates formation of MC1 Tau. However, in a hyperphosphorylating environment, the formation of MC1 was abrogated, but Hsc70 binding to Tau was enhanced. Thus, under normal circumstances, MC1 formation may be a protective conformation facilitated by Hsc70. However, in a diseased environment, Hsc70 may preserve Tau in a more unstructured state, perhaps facilitating its pathogenicity.