Showing papers by "Jason E. Gestwicki published in 2010"

Heat shock protein 70 (hsp70) as an emerging drug target.

Abstract: Heat shock protein 70 (Hsp70) is a molecular chaperone that is expressed in response to stress. In this role, Hsp70 binds to its protein substrates and stabilize them against denaturation or aggregation until conditions improve.1 In addition to its functions during a stress response, Hsp70 has multiple responsibilities during normal growth; it assists in the folding of newly synthesized proteins,2, 3 the subcellular transport of proteins and vesicles,4 the formation and dissociation of complexes,5 and the degradation of unwanted proteins.6, 7 Thus, this chaperone broadly shapes protein homeostasis by controlling protein quality control and turnover during both normal and stress conditions.8 Consistent with these diverse activities, genetic and biochemical studies have implicated it in a range of diseases, including cancer, neurodegeneration, allograft rejection and infection. This review provides a brief review of Hsp70 structure and function and then explores some of the emerging opportunities (and challenges) for drug discovery. Hsp70 is Highly Conserved Members of the Hsp70 family are ubiquitously expressed and highly conserved; for example, the major Hsp70 from Escherichia coli, termed DnaK, is approximately 50% identical to human Hsp70s.9 Eukaryotes often express multiple Hsp70 family members with major isoforms found in all the cellular compartments: Hsp72 (HSPA1A) and heat shock cognate 70 (Hsc70/HSPA8) in the cytosol and nucleus, BiP (Grp78/HSPA5) in the endoplasmic reticulum and mtHsp70 (Grp75/mortalin/HSPA9) in mitochondria. Some of the functions of the cytosolic isoforms, Hsc70 and Hsp72, are thought to be redundant, but the transcription of Hsp72 is highly responsive to stress and Hsc70 is constitutively expressed. In the ER and mitochondria, the Hsp70 family members are thought to fulfill specific functions and have unique substrates, with BiP playing key roles in the folding and quality control of ER proteins and mtHsp70 being involved in the import and export of proteins from the mitochondria. For the purposes of this review, we will often use Hsp70 as a generic term to encompass the shared properties of the family members.

Phenothiazine-mediated rescue of cognition in tau transgenic mice requires neuroprotection and reduced soluble tau burden.

Abstract: Background: It has traditionally been thought that the pathological accumulation of tau in Alzheimer’s disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection. Results: Here, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember™, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced. Conclusions: Neuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial “magic bullet” because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.

Binding of a small molecule at a protein-protein interface regulates the chaperone activity of hsp70-hsp40.

Abstract: Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70-Hsp40 complex assembly at a native protein-protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis.

High-Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume by Exploiting Energy Transfer

Abstract: 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.

Conditionally controlling nuclear trafficking in yeast by chemical-induced protein dimerization

Abstract: We present here a protocol to conditionally control the nuclear trafficking of target proteins in yeast. In this system, rapamycin is used to heterodimerize two chimeric proteins. One chimera consists of a FK506-binding protein (FKBP12) fused to a cellular 'address' (nuclear localization signal or nuclear export sequence). The second chimera consists of a target protein fused to a fluorescent protein and the FKBP12-rapamycin-binding (FRB) domain from FKBP-12-rapamycin associated protein 1 (FRAP1, also known as mTor). Rapamycin induces dimerization of the FKBP12- and FRB-containing chimeras; these interactions selectively place the target protein under control of the cell address, thereby directing the protein into or out of the nucleus. By chemical-induced dimerization, protein mislocalization is reversible and enables the identification of conditional loss-of-function and gain-of-function phenotypes, in contrast to other systems that require permanent modification of the targeted protein. Yeast strains for this analysis can be constructed in 1 week, and the technique allows protein mislocalization within 15 min after drug treatment.

Chemical probes that selectively recognize the earliest Aβ oligomers in complex mixtures.

Abstract: Alzheimer's disease (AD) is characterized by the self-assembly of amyloid beta (Aβ) peptides. Recent models implicate some of the earliest Aβ oligomers, such as trimers and tetramers, in disease. However, the roles of these structures remain uncertain, in part, because selective probes of their formation are not available. Toward that goal, we generated bivalent versions of the known Aβ ligand, the pentapeptide KLVFF. We found that compounds containing sufficiently long linkers (∼19 to 24 A) recognized primarily Aβ trimers and tetramers, with little binding to either monomer or higher order structures. These compounds might be useful probes for early Aβ oligomers.

Quantifying Prefibrillar Amyloids in vitro by Using a “Thioflavin-Like” Spectroscopic Method

Abstract: In Alzheimer's disease (AD) and other neurodegenerative disorders, proteins accumulate into ordered aggregates, called amyloids. Recent evidence suggests that these structures include both large, insoluble fibrils and smaller, prefibrillar structures, such as dimers, oligomers, and protofibrils. Recently, focus has shifted to the prefibrillar aggregates because they are highly neurotoxic and their levels appear to correlate with cognitive impairment. Thus, there is interest in finding methods for specifically quantifying these structures. One of the classic ways of detecting amyloid formation is through the fluorescence of the benzothiazole dye, thioflavin T (ThT). This reagent has been a "workhorse" of the amyloid field because it is robust and inexpensive. However, one of its limitations is that it does not distinguish between prefibrillar and fibrillar aggregates. We screened a library of 37 indoles for those that selectively change fluorescence in the presence of prefibrillar amyloid-beta (Abeta). From this process, we selected the most promising example, tryptophanol (TROL), to use in a quantitative "thioflavin-like" assay. Using this probe in combination with electron microscopy, we found that prefibrils are largely depleted during Abeta aggregation in vitro but that they remain present after the apparent saturation of the ThT signal. These results suggest that a combination of TROL and ThT provides greater insight into the process of amyloid formation by Abeta. In addition, we found that TROL also recognizes other amyloid-prone proteins, including ataxin-3, amylin, and CsgA. Thus, this assay might be an inexpensive spectroscopic method for quantifying amyloid prefibrils in vitro.

Analytical system with photonic crystal sensor

Abstract: A system for determining whether interaction occurs between a trial substance and a target substance. The system includes a photonic crystal sensor having a photonic crystal structure and a defect member disposed adjacent the photonic crystal structure. The defect member defines an operative surface able to receive the target substance and the trial substance. The system further includes a light source that inputs a light signal to the photonic crystal structure and the defect member. The light signal is internally reflected, and a resultant output signal is outputted. The output signal relates to whether the trial substance interacts with the target substance at the operative surface. Furthermore, the system includes an identity detector that identifies the trial substance that interacts with the target substance.

Enantioselective Organocatalytic Hantzsch Synthesis of Polyhydroquinolines.

Abstract: The following changes were made to the revised Supporting Information document submitted with this correction: (1) All data referring to compound 4o has been removed. (2) HPLC traces for 4a, 4d, 4h, and 4n have been replaced with less ambiguous choices, pp 43−47. (3) Clarification of the method to confirm peak identities is now included, p 48. (4) Confirmation of peak identities is now included for compounds 4a, 4d, 4h, 4k, 4l, and 4n, pp 48−50. (5) Missing retention times for compounds 4a and 4g have been added on p 42. (6) H NMR peaks have been added for compounds 4k and 4j. These were missing in the original document, p 5. (7) The new Supplemental Figure 1 has been added (p 2), which shows that the Hantzsch reaction to produce compound 4b does not proceed in the absence of catalyst. We previously neglected to include this important data. This material is available free of charge via the Internet at http://pubs.acs.org.

Special series: Molecular chaperones in protein folding and disease

Abstract: P ioneering work by Anfinsen established that the folding of a polypeptide is dictated by its primary sequence. The study of folding remains an active area of research and a question of emerging importance is how the special challenges imposed by the cytoplasm (e.g., high protein load, molecular crowding, and dynamic oligomerization) impact this process. Given the opportunities for off-pathway interactions and the threat of catastrophic aggregation, the cellular environment certainly offers unique challenges. To overcome these difficulties, cells express molecular chaperones, a family of abundant, evolutionarily conserved proteins that directly bind to protein substrates. These interactions have many consequences, including more efficient folding of nascent polypeptides and stabilization of the folding trajectory. However, chaperones also control many other aspects of a protein’s life and death, including effects on aggregation, assembly into multiprotein complexes, subcellular transport, and degradation. In these various tasks, some chaperones appear to interact with a large percentage of the proteome, whereas others bind to a more restricted selection of substrates, for example, only specific components of chromatin or the proteasome. Of the molecular chaperones, the heat shock proteins are a powerful defense against environmental insults, such as thermal or oxidative stress. The heat shock proteins are typically categorized by their average molecular weight, with the major classes being Hsp40, Hsp60, Hsp70, Hsp90, Hsp104, Hsp110, and the small heat shock proteins. In the cell, members of these categories appear to play distinct roles, and consequently, they exhibit little structural or sequence homology. However, in response to stress, they share the feature of markedly increased expression. Moreover, many of the heat shock proteins are thought to work together toward the goal of shaping the proteome. For example, Hsp70 and Hsp90 cooperate in the folding and trafficking of nuclear hormone receptors, and the function of many Hsp40s is thought to occur via their actions on Hsp70. In addition, it is becoming increasingly evident that the heat shock proteins are integrated with the ubiquitin-proteasome system, endoplasmic reticulum-associated degradation, the unfolded protein response, apoptosis signaling, the chaperone-mediated autophagy pathway and other cellular quality control systems. Collectively, these systems describe a proteostasis network,9 which regulates cellular fate through managing proteome integrity. Understanding the logic of this system will likely require more detailed knowledge of how the components are interconnected in both cells and organisms. Toward this goal, attempts to mathematically model the chaperone network are an important addition10,11 and unbiased interaction maps should also be informative. Beyond their roles in fundamental protein homeostasis, molecular chaperones are emerging drug targets. Many diseases are characterized by disregulation of proteostasis, including the classic examples of protein misfolding disorders, such as cystic fibrosis and Alzheimer’s disease. Despite a clear role for aberrant protein processing in these disorders, the path to therapeutic rescue of folding balance remains uncertain. One expected benefit of a deeper understanding of chaperone biology is the ability to predict new drug targets and strategies. In support of this idea, inhibitors of the Hsp90 chaperone are currently being explored in multiple oncology clinical trials. Inhibition of Hsp90 has been shown to reduce the stability of prosurvival substrates, resulting in apoptosis of susceptible cells. It seems likely that other nodes within the cellular quality control network will also prove to be amenable to pharmacological targeting. However, chaperones often act on a large subset of the proteome, which means that achieving the desired therapeutic outcomes without concurrent toxicity will involve a delicate balance. In contrast, chemicals that add folding energy to specific misfolding-prone targets have also been explored in protein misfolding diseases. Although these strategies involve more selectivity, chemical optimization is required for each target that generates a distinct set of design challenges. It seems that a combination of strategies will likely be the best long-term scenario. In this Special Series, experts in the field provide comprehensive updates on the most recent developments in the study of molecular chaperones, protein folding, and disease. In navigating this Special Series, one important question to consider is: what are some major, current goals of the field? Editorial