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1.  High-Throughput Screen of Natural Product Extracts in A Yeast Model of Polyglutamine Proteotoxicity 
Chemical biology & drug design  2014;83(4):440-449.
Proteins with expanded polyglutamine (polyQ) segments cause a number of fatal neurodegenerative disorders, including Huntington’s disease (HD). Previous high-throughput screens in cellular and biochemical models of HD have revealed compounds that mitigate polyQ aggregation and proteotoxicity, providing insight into the mechanisms of disease and leads for potential therapeutics. However, the structural diversity of natural products has not yet been fully mobilized toward these goals. Here, we have screened a collection of ~11 000 natural product extracts for the ability to recover the slow growth of ΔProQ103-expressing yeast cells in 384-well plates (Z’ ~ 0.7, CV ~ 8%). This screen identified actinomycin D as a strong inhibitor of polyQ aggregation and proteotoxicity at nanomolar concentrations (~50–500 ng/mL). We found that a low dose of actinomycin D increased the levels of the heat-shock proteins Hsp104, Hsp70 and Hsp26 and enhanced binding of Hsp70 to the polyQ in yeast. Actinomycin also suppressed aggregation of polyQ in mammalian cells, suggesting a conserved mechanism. These results establish natural products as a rich source of compounds with interesting mechanisms of action against polyQ disorders.
doi:10.1111/cbdd.12259
PMCID: PMC4068144  PMID: 24636344
heat shock protein 70; high throughput screening; Huntington’s disease; molecular chaperones
2.  Direct and Propagated Effects of Small Molecules on Protein–Protein Interaction Networks 
Networks of protein–protein interactions (PPIs) link all aspects of cellular biology. Dysfunction in the assembly or dynamics of PPI networks is a hallmark of human disease, and as such, there is growing interest in the discovery of small molecules that either promote or inhibit PPIs. PPIs were once considered undruggable because of their relatively large buried surface areas and difficult topologies. Despite these challenges, recent advances in chemical screening methodologies, combined with improvements in structural and computational biology have made some of these targets more tractable. In this review, we highlight developments that have opened the door to potent chemical modulators. We focus on how allostery is being used to produce surprisingly robust changes in PPIs, even for the most challenging targets. We also discuss how interfering with one PPI can propagate changes through the broader web of interactions. Through this analysis, it is becoming clear that a combination of direct and propagated effects on PPI networks is ultimately how small molecules re-shape biology.
doi:10.3389/fbioe.2015.00119
PMCID: PMC4547496  PMID: 26380257
multi-protein complexes; allostery; PPI inhibition
3.  Targeting Proteostasis Through the Protein Quality Control Function of the Hsp90/Hsp70-based Chaperone Machinery for Treatment of Adult Onset Neurodegenerative Diseases 
Currently available therapies for adult onset neurodegenerative diseases provide symptomatic relief, but are not disease modifying. We explore here a new neuroprotective approach based on drugs targeting chaperone-directed protein quality control. Critical target proteins that unfold and aggregate in these diseases, such as the polylglutamine androgen receptor (spinal and bulbar muscular atrophy), huntingtin (Huntington’s disease), α-synuclein (Parkinson’s disease) and tau (Alzheimer’s disease) are client proteins of Hsp90, and their turnover is regulated by the protein quality control function of the Hsp90/Hsp70-based chaperone machinery. In protein quality control Hsp90 and Hsp70 have opposing effects on client protein stability; Hsp90 stabilizes the clients and inhibits their ubiquitination, whereas Hsp70 promotes CHIP-dependent ubiquitination and proteasomal degradation. We discuss how drugs that modulate proteostasis by inhibiting Hsp90 function or by promoting Hsp70 function enhance the degradation of the critical aggregating proteins and ameliorate toxic symptoms in cell and animal disease models.
doi:10.1146/annurev-pharmtox-010814-124332
PMCID: PMC4372135  PMID: 25292434
Neurodegeneration; protein aggregation; ubiquitination; CHIP; proteasome
4.  Expanding the Number of “Druggable” Targets: Non-Enzymes and Protein-Protein Interactions 
Chemical biology & drug design  2013;81(1):22-32.
Following sequencing and assembly of the human genome, the preferred methods for identification of new drug targets have changed dramatically. Modern tactics such as genome-wide association studies (GWAS) and deep sequencing are fundamentally different from the pharmacology-guided approaches used previously, in which knowledge of small molecule ligands acting at their cellular targets was the primary discovery engine. A consequence of the “target-first, pharmacology-second” strategy is that many predicted drug targets are non-enzymes, such as scaffolding, regulatory or structural proteins, and their activities are often dependent on protein-protein interactions (PPIs). These types of targets create unique challenges to drug discovery efforts because enzymatic turnover cannot be used as a convenient surrogate for compound potency. Moreover, it is often challenging to predict how ligand binding to non-enzymes might affect changes in protein function and/or pathobiology. Thus, in the post-genomic era, targets might be strongly implicated by molecular biology-based methods, yet they often later earn the designation of “undruggable.” Can the scope of available targets be widened to include these promising, but challenging, non-enzymes? In this review, we discuss advances in high throughput screening technology and chemical library design that are emerging to deal with these challenges.
doi:10.1111/cbdd.12066
PMCID: PMC3531880  PMID: 23253128
5.  Hsp70 Protein Complexes as Drug Targets 
Current pharmaceutical design  2013;19(3):404-417.
Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70’s interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, such as pro-folding, pro-degradation and pro-trafficking. Thus, a promising strategy may be to block protein-protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to those goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology.
PMCID: PMC3593251  PMID: 22920901
heat shock protein 70; molecular chaperones; J proteins; nucleotide exchange factors; tetratricopeptide repeat-containing proteins; protein complex
6.  Cysteine Reactivity Distinguishes Redox Sensing by the Heat Inducible and Constitutive Forms of Heat Shock Protein 70 (Hsp70) 
Chemistry & biology  2012;19(11):1391-1399.
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 over-expression of the C306S mutant blocked MB-mediated loss of tau in a cellular model. Further, 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 over-expression 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.
doi:10.1016/j.chembiol.2012.07.026
PMCID: PMC3508472  PMID: 23177194
7.  A Model in which Hsp90 Targets Protein Folding Clefts: rationale for a New Approach to Neuroprotective Treatment of Protein Folding Diseases 
In an EBM Minireview published in 2010, we proposed that the Hsp90/Hsp70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation (1). The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand binding clefts (2). The model provides a framework for thinking about the development of neuroprotective therapies for protein folding diseases like Alzheimer’s disease (AD), Parkinson’s disease (PD) and the polyglutamine expansion disorders, such as Huntington’s disease (HD) and spinal and bulbar muscular atrophy (SBMA). Major aberrant proteins that misfold and accumulate in these diseases are ‘client’ proteins of the abundant and ubiquitous stress chaperone Hsp90 (3). These Hsp90 client proteins include tau (AD), α-synuclein (PD), huntingtin (HD) and the expanded glutamine androgen receptor (polyQ AR) (SBMA). In this minireview we update our model in which Hsp90 acts on protein folding clefts and show how it forms a rational basis for developing drugs that promote the targeted elimination of these aberrant proteins.
doi:10.1177/1535370214539444
PMCID: PMC4318483  PMID: 24990484
8.  Synthetic lethal interactions in yeast reveal functional roles of J protein co-chaperones 
Molecular bioSystems  2012;8(11):2901-2908.
J proteins are a diverse family of co-chaperones that cooperate with heat shock protein 70 (Hsp70) to coordinate protein quality control, especially in response to cellular stress. Current models suggest that individual J proteins might play roles in recruiting Hsp70s to specific functions, such as maintaining cell wall integrity or promoting ribosome biogenesis. However, relatively few stresses have been used to test this model and, as a result, only a few specific activities have been identified. To expand our understanding of the J protein network, we used a synthetic lethal approach in which 11 Saccharomyces cerevisiae deletion strains were treated with 12 well-characterized chemical inhibitors. The results defined new roles for specific J proteins in major signaling pathways. For example, an important role for Swa2 in cell wall integrity was identified and activities of the under-explored Jjj1, Apj1, Jjj3 and Caj1 proteins were suggested. More generally, these findings support a model in which some J proteins, such as Ydj1 and Zuo1, play “generalist” roles, while others, such as Apj1 and Jjj2, are “specialists”, having roles in relatively few pathways. Together, these results provide new insight into the network of J proteins.
doi:10.1039/c2mb25248a
PMCID: PMC3463740  PMID: 22851130
9.  Analysis of the Tau-Associated Proteome Reveals that Exchange of Hsp70 for Hsp90 Is Involved in Tau Degradation 
ACS chemical biology  2012;7(10):1677-1686.
The microtubule associated protein tau (MAPT/tau) aberrantly accumulates in fifteen neurodegenerative diseases, termed tauopathies. One way to treat tauopathies may be to accelerate tau clearance, but the molecular mechanisms governing tau stability are not yet clear. We recently identified chemical probes that markedly accelerate the clearance of tau in cellular and animal models. In the current study, we used one of these probes in combination with immunoprecipitation and mass spectrometry to identify 48 proteins whose association with tau changes during the first 10 minutes after treatment. These proteins included known modifiers of tau proteotoxicity, such as ILF-2 (NFAT), ILF-3, and ataxin-2. A striking observation from the dataset was that tau binding to heat shock protein 70 (Hsp70) decreased while binding to Hsp90 significantly increased. Both chaperones have been linked to tau homeostasis, but their mechanisms have not been established. Using peptide arrays and binding assays, we found that Hsp70 and Hsp90 appeared to compete for binding to shared sites on tau. Further, the Hsp90-bound complex proved to be important in initiating tau clearance in cells. These results suggest that the relative levels of Hsp70 and Hsp90 may help determine whether tau is retained or degraded. Consistent with this model, analysis of reported microarray expression data from Alzheimer’s disease patients and age-matched controls showed that the levels of Hsp90 are reduced in the diseased hippocampus. These studies suggest that Hsp70 and Hsp90 work together to coordinate tau homeostasis.
doi:10.1021/cb3002599
PMCID: PMC3477299  PMID: 22769591
10.  Pharmacological Tuning of Heat Shock Protein 70 Modulates Polyglutamine Toxicity and Aggregation 
ACS chemical biology  2012;7(9):1556-1564.
Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70’s ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify Hsp70’s role as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.
doi:10.1021/cb300166p
PMCID: PMC3448832  PMID: 22709427
polyQ; protein misfolding; molecular chaperones; heat shock protein 70 (Hsp70); proteostasis; chemical genetics; chemical probes
11.  MOLECULAR CHAPERONES DNAK AND DNAJ SHARE PREDICTED BINDING SITES ON MOST PROTEINS IN THE E. COLI PROTEOME 
Molecular bioSystems  2012;8(9):2323-2333.
In Escherichia coli, the molecular chaperones DnaK and DnaJ cooperate to assist the folding of newly synthesized or unfolded polypeptides. DnaK and DnaJ bind to hydrophobic motifs in these proteins and also each other to promote folding. This system is thought to be sufficiently versatile to act on the entire proteome, which creates interesting challenges in understanding the large-scale, ternary interactions between DnaK, DnaJ and their thousands of potential substrates. To address this question, we computationally predicted the number and frequency of DnaK- and DnaJ-binding motifs in the E. coli proteome, guided by free energy-based binding consensus motifs. This analysis revealed that nearly every protein is predicted to contain multiple DnaK- and DnaJ-binding sites, with the DnaJ sites occurring approximately twice as often. Further, we found that an overwhelming majority of the DnaK sites partially or completely overlapped with the DnaJ-binding motifs. It is well known that high concentrations of DnaJ inhibit DnaK-DnaJ-mediated refolding. The observed overlapping binding sites suggest that this phenomenon may be explained by an important balance in the relative stoichiometry of DnaK and DnaJ which determines whether they bind synergistically or competitively. To test this idea, we measured the chaperone-assisted folding of two denatured substrates and found that the distribution of predicted DnaK- and DnaJ-binding sites was indeed a good predictor of the optimal stoichiometry required for folding. These studies provide insight into how DnaK and DnaJ might cooperate to maintain global protein homeostasis.
doi:10.1039/c2mb25145k
PMCID: PMC3462289  PMID: 22732719
12.  Features of Protein-Protein Interactions that Translate into Potent Inhibitors: Topology, Surface Area and Affinity 
Protein-protein interactions (PPIs) control the assembly of multi-protein complexes and, thus, these contacts have enormous potential as drug targets. However, the field has produced a mix of both exciting success stories and frustrating challenges. Here, we review known examples and explore how the physical features of a PPI, such as its affinity, hotspots, off-rates, buried surface area and topology, may influence the chances of success in finding inhibitors. This analysis suggests that concise, tight binding PPIs are most amenable to inhibition. However, it is also clear that emerging technical methods are expanding the repertoire of “druggable” protein contacts and increasing the odds against difficult targets. In particular, natural product-like compound libraries, high throughput screens specifically designed for PPIs and approaches that favor discovery of allosteric inhibitors appear to be attractive routes. The first group of PPI inhibitors has entered clinical trials, further motivating the need to understand the challenges and opportunities in pursuing these types of targets.
doi:10.1017/erm.2012.10
PMCID: PMC3591511  PMID: 22831787
high throughput screening; allostery; multi-protein complexes; Hsp70; Hsp90; fragment based drug discovery; natural products; protein-protein interactions
13.  An Allosteric Modulator of HIV-1 Protease Shows Equipotent Inhibition of Wild-Type and Drug-Resistant Proteases 
Journal of Medicinal Chemistry  2014;57(15):6468-6478.
NMR and MD simulations have demonstrated that the flaps of HIV-1 protease (HIV-1p) adopt a range of conformations that are coupled with its enzymatic activity. Previously, a model was created for an allosteric site located between the flap and the core of HIV-1p, called the Eye site (Biopolymers2008, 89, 643−65218381626). Here, results from our first study were combined with a ligand-based, lead-hopping method to identify a novel compound (NIT). NIT inhibits HIV-1p, independent of the presence of an active-site inhibitor such as pepstatin A. Assays showed that NIT acts on an allosteric site other than the dimerization interface. MD simulations of the ligand–protein complex show that NIT stably binds in the Eye site and restricts the flaps. That bound state of NIT is consistent with a crystal structure of similar fragments bound in the Eye site (Chem. Biol. Drug Des.2010, 75, 257−26820659109). Most importantly, NIT is equally potent against wild-type and a multidrug-resistant mutant of HIV-1p, which highlights the promise of allosteric inhibitors circumventing existing clinical resistance.
doi:10.1021/jm5008352
PMCID: PMC4136727  PMID: 25062388
14.  Allosteric drugs: the interaction of anti-tumor compound MKT-077 with human Hsp70 chaperones 
Journal of molecular biology  2011;411(3):614-632.
The Hsp70 chaperones (Heat shock protein 70 kDa) are key to cellular protein homeostatis. 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 are increasingly been 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 anti-cancer compound MKT-077, is through differential interaction with the Hsp70 allosteric states. MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4- oxothiazolidin-2-ylidenemethyl] pyridinium chloride) is therefore an “allosteric drug”. Using NMR spectroscopy, the compound’s binding site on human HSPA8 (Hsc70) is identified. The binding pose is obtained from NMR-restrained docking calculations, subsequently scored by molecular dynamics-based energy and solvation computations. Suggestions for improvement of the compound’s properties are made on the basis of the binding location and pose.
doi:10.1016/j.jmb.2011.06.003
PMCID: PMC3146629  PMID: 21708173
15.  Insight into Amyloid Structure Using Chemical Probes 
Chemical biology & drug design  2011;77(6):399-411.
Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by the deposition of amyloids in the brain. One prominent form of amyloid is composed of repeating units of the amyloid-β (Aβ) peptide. Over the past decade, it has become clear that these Aβ amyloids are not homogeneous; rather, they are composed of a series of structures varying in their overall size and shape and the number of Aβ peptides they contain. Recent theories suggest that these different amyloid conformations may play distinct roles in disease, although their relative contributions are still being discovered. Here, we review how chemical probes, such as congo red, thioflavin T and their derivatives, have been powerful tools for better understanding amyloid structure and function. Moreover, we discuss how design and deployment of conformationally selective probes might be used to test emerging models of AD.
doi:10.1111/j.1747-0285.2011.01110.x
PMCID: PMC3097318  PMID: 21457473
Alzheimer’s disease; thioflavin T; congo red; curcumin; fibrils; protofibrils; oligomers; amyloid beta
16.  Chemical Screens Against A Reconstituted Multi-Protein Complex: Myricetin Blocks DnaJ Regulation of DnaK through an Allosteric Mechanism 
Chemistry & biology  2011;18(2):210-221.
SUMMARY
DnaK is a molecular chaperone responsible for multiple aspects of 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 re-constituted 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 DnaJ. Together, these results highlight a “gray box” screening approach, which approximates a limited amount of the complexity expected in physiological, multi-protein systems.
doi:10.1016/j.chembiol.2010.12.010
PMCID: PMC3057461  PMID: 21338918
17.  High Throughput Screen for Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK): ATPase Assay in Low Volume By Exploiting Energy Transfer 
Journal of biomolecular screening  2010;15(10):1211-1219.
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). While 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, we 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, we tested 55,400 compounds against DnaK, a prokaryotic member of the Hsp70 family. The assay performance was good (Z′ ~ 0.6, CV ~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.
doi:10.1177/1087057110380571
PMCID: PMC3052282  PMID: 20926844
phosphate; malachite green; ATPase; molecular chaperone; fluorescence assay
18.  Heat Shock Protein 70 (Hsp70) as an Emerging Drug Target 
Journal of medicinal chemistry  2010;53(12):4585-4602.
doi:10.1021/jm100054f
PMCID: PMC2895966  PMID: 20334364
19.  A Chemical Screening Approach Reveals that Indole Flourescence is Quenched by Pre-Fibrillar But Not Fibrillar Amyloid-β 
Aggregated amyloid-β (Aβ) peptide is implicated in the pathology of Alzheimer’s disease. In vitro and in vivo, these aggregates are found in a variety of morphologies, including globular oligomers and linear fibrils, which possess distinct biological activities. However, known chemical probes, including the dyes thioflavin T and Congo Red, appear to lack selectivity for specific amyloid structures. To identify molecules that might differentiate between these architectures, we employed a fluorescence-based interaction assay to screen a collection of 68 known Aβ ligands against pre-formed oligomers and fibrils. In these studies, we found that the fluorescence of five indole-based compounds was selectively quenched (~15%) in the presence of oligomers, but remained unchanged after addition of fibrils. These results suggest that indoles might be complementary to existing chemical probes for studying amyloid formation in vitro.
doi:10.1016/j.bmcl.2009.07.082
PMCID: PMC2730169  PMID: 19640715
Alzheimer’s disease; neurodegeneration; protein misfolding; thioflavin T; fluorescence
20.  Synthesis of Orthogonally Reactive FK506 Derivatives via Olefin Cross Metathesis 
Bioorganic & medicinal chemistry  2009;17(16):5763-5768.
Chemical inducers of dimerization (CIDs) are employed in a wide range of biological applications, to control protein localization, modulate protein-protein interactions and improve drug lifetimes. These bifunctional chemical probes are assembled from two synthetic modules, which each provide affinity for a distinct protein target. FK506 and its derivatives are often employed as modules in the syntheses of these bifunctional constructs, owing to the abundance and favorable distribution of their target, FK506-binding protein (FKBP). However, the structural complexity of FK506 necessitates multi-step syntheses and/or multiple protection-deprotection schemes prior to installation into CIDs. In this work, we describe an efficient, one-step synthesis of FK506 derivatives through a selective, microwave-accelerated, cross metathesis diversification step of the C39 terminal alkene. Using this approach, FK506 is modified with an array of functional groups, including primary amines and carboxylic acids, which make the resulting derivatives suitable for the modular assembly of CIDs. To illustrate this idea, we report the synthesis of a heterobifunctional HIV protease inhibitor.
doi:10.1016/j.bmc.2009.07.030
PMCID: PMC2758530  PMID: 19643614
metathesis; cross-coupling; natural products; chemical inducers of dimerization; drug targeting; HIV protease
21.  Pharmacological Targeting of the Hsp70 Chaperone 
The molecular chaperone, heat shock protein 70 (Hsp70), acts at multiple steps in a protein’s life cycle, including during the processes of folding, trafficking, remodeling and degradation. To accomplish these various tasks, the activity of Hsp70 is shaped by a host of co-chaperones, which bind to the core chaperone and influence its functions. Genetic studies have strongly linked Hsp70 and its co-chaperones to numerous diseases, including cancer, neurodegeneration and microbial pathogenesis, yet the potential of this chaperone as a therapeutic target remains largely underexplored. Here, we review the current state of Hsp70 as a drug target, with a special emphasis on the important challenges and opportunities imposed by its co-chaperones, protein-protein interactions and allostery.
PMCID: PMC2799686  PMID: 19860737
proteostasis; flavonoids; dihydropyrimidines; spergualin; sulfoglycolipids; geranylgeranyl acetone; protein folding; ATPase; protein-protein interactions
22.  Analogues of the Allosteric Heat Shock Protein 70 (Hsp70) Inhibitor, MKT-077, As Anti-Cancer Agents 
ACS Medicinal Chemistry Letters  2013;4(11):1042-1047.
The rhodacyanine, MKT-077, has antiproliferative activity against cancer cell lines through its ability to inhibit members of the heat shock protein 70 (Hsp70) family of molecular chaperones. However, MKT-077 is rapidly metabolized, which limits its use as either a chemical probe or potential therapeutic. We report the synthesis and characterization of MKT-077 analogues designed for greater stability. The most potent molecules, such as 30 (JG-98), were at least 3-fold more active than MKT-077 against the breast cancer cell lines MDA-MB-231 and MCF-7 (EC50 values of 0.4 ± 0.03 and 0.7 ± 0.2 μM, respectively). The analogues modestly destabilized the chaperone clients, Akt1 and Raf1, and induced apoptosis in these cells. Further, the microsomal half-life of JG-98 was improved at least 7-fold (t1/2 = 37 min) compared to MKT-077 (t1/2 < 5 min). Finally, NMR titration experiments suggested that these analogues bind an allosteric site that is known to accommodate MKT-077. These studies advance MKT-077 analogues as chemical probes for studying Hsp70s roles in cancer.
doi:10.1021/ml400204n
PMCID: PMC3845967  PMID: 24312699
Breast cancer; mortalin; Hsp90; proteostasis; p53
23.  Analogs of the Allosteric Heat Shock Protein 70 (Hsp70) Inhibitor, MKT-077, as Anti-Cancer Agents 
ACS medicinal chemistry letters  2013;4(11):10.1021/ml400204n.
The rhodacyanine, MKT-077, has anti-proliferative activity against cancer cell lines through its ability to inhibit members of the heat shock protein 70 (Hsp70) family of molecular chaperones. However, MKT-077 is rapidly metabolized, which limits its use as either a chemical probe or potential therapeutic. We report the synthesis and characterization of MKT-077 analogs designed for greater stability. The most potent molecules, such as 30 (JG-98), were at least 3-fold more active than MKT-077 against the breast cancer cell lines MDA-MB-231 and MCF-7 (EC50 values of 0.4 ± 0.03 μM and 0.7 ± 0.2 μM, respectively). The analogs modestly destabilized the chaperone “clients”, Akt1 and Raf1, and induced apoptosis in these cells. Further, the microsomal half-life of JG-98 was improved at least 7-fold (t1/2 = 37 min) compared to MKT-077 (t1/2 < 5 min). Finally, NMR titration experiments suggested that these analogs bind an allosteric site that is known to accommodate MKT-077. These studies advance MKT-077 analogs as chemical probes for studying Hsp70’s roles in cancer.
doi:10.1021/ml400204n
PMCID: PMC3845967  PMID: 24312699
breast cancer; mortalin; Hsp90; proteostasis; p53
24.  Development of a Capillary Electrophoresis Platform for Identifying Inhibitors of Protein-Protein Interactions 
Analytical chemistry  2013;85(20):10.1021/ac4023082.
Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound to free ratio. The method was used to screen a library of 3,443 compounds and results compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that reconfirmed in subsequent testing suggesting greater specificity. This finding was attributed to use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens but at the current stage of development it is attractive as a secondary screen to test hits found by higher throughput methods.
doi:10.1021/ac4023082
PMCID: PMC3845662  PMID: 24060167
25.  Inhibitors of Difficult Protein-Protein Interactions Identified by High Throughput Screening of Multi-Protein Complexes 
ACS chemical biology  2013;8(9):1988-1997.
Protein-protein interactions (PPIs) are important in all aspects of cellular function and there is interest in finding inhibitors of these contacts. However, PPIs with weak affinities and/or large interfaces have traditionally been more resistant to the discovery of inhibitors, partly because it is more challenging to develop high throughput screening (HTS) methods that permit direct measurements of these physical interactions. Here, we explored whether the functional consequences of a weak PPI might be used as a surrogate for binding. As a model, we used the bacterial ATPase DnaK and its partners DnaJ and GrpE. Both DnaJ and GrpE bind DnaK and catalytically accelerate its ATP cycling, so we used stimulated nucleotide turnover to indirectly report on these PPIs. In pilot screens, we identified compounds that block activation of DnaK by either DnaJ or GrpE. Interestingly, at least one of these molecules blocked binding of DnaK to DnaJ, while another compound disrupted allostery between DnaK and GrpE without altering the physical interaction. These findings suggest that the activity of a reconstituted multi-protein complex might be used in some cases to identify allosteric inhibitors of challenging PPIs.
doi:10.1021/cb400356m
PMCID: PMC3783524  PMID: 23819499
allostery; molecular chaperones; co-chaperone; ATPase assay; Hsp70; Hsp40

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