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A hydraulic control unit system with a mechanical oil pump (MOP) and an electric oil pump (EOP) was newly designed for the lubrication and clutch-to-clutch control of a two-speed dedicated electric transmission for battery electric vehicles (BEVs), in order to solve the high-cost, poor-noise and high-energy-consumption problem exposed in previous single-EOP design with big displacement. The driving motor for BEVs can’t idle like the combustion engine when the vehicle stops still, so an EOP with small displacement is required to supply enough fluid to start the vehicle except to a low-cost MOP. Four control algorithms for the two-pump system were developed as well as the main line pressure control strategy, which were confirmed by a Hardware-in-the-Loop (HIL) test bench. The simulated and tested total pump energy consumption in a driving circle WLTC shows that the equal-power control algorithm and the transient on-demand control algorithm can obtain better performance for saving energy consumption. Several oil displacement parameter groups for the MOP and the EOP are discussed, and the results show that proper displacement group for the EOP and the MOP can obtain good balance for the total cost and the performance with the combination of proper two-pump control strategies.

A copper-mediated synthesis of diaryl sulfides utilizing Cu(I)-thiophene-2-carboxylate (CuTC) is described. We demonstrate the use of CuTC as a soluble, non-basic catalyst in the coupling of aryl iodides and aryl thiols in the synthesis of synthetically advanced diaryl sulfides. This method allows for the successful coupling of challenging substrates including ortho-substituted and heteroaryl iodides and thiols. Additionally, most of the aryl iodide substrates used here contain the privileged piperazine scaffold bound to a pyrimidine, pyridine, or phenyl ring and thus this method allows for the elaboration of complex piperazine scaffolds into molecules of biological interest. The method described here enables the incorporation of late-stage structural diversity into diaryl sulfides containing the piperazine ring, thus enhancing the number and nature of derivatives available for SAR investigation.

The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.

SummaryHsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70.

Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.

Hsp90 chaperones play a critical role in modulating the activity of many cell signaling proteins and are an attractive target for anti-cancer therapeutics. We report here the structures of the water soluble 8-aryl-sulfanyl adenine class Hsp90 inhibitors, 1 (PU-H71) and 2 (PU-H64), in complex with the N-terminal domain of human Hsp90alpha. The conformation of 1 when bound to Hsp90 differs from previously reported 8-aryl adenine Hsp90 inhibitors including 3 (PU24FCl). While the binding mode for 3 places the 2'-halide of the 8-aryl group on top of the adenine ring, for 1 and 2, we show that the 2'-halide is rotated approximately 180 degrees away. This difference explains the opposing trends in Hsp90 inhibitory activity for the 2'-halo derivatives of the 3',4',5'-trimethoxy series where ClBrI compared to the 4',5'-methylenedioxy series where IBrCl. We also present quantum chemical calculations of 2 and its analogues that illuminate their basis for Hsp90 inhibition. The calculated conformation of 2 agreed well with the crystallographically observed conformations of 1 and 2. The predictive nature of the calculations has allowed the exploration of additional derivatives based on the 8-aryl adenine scaffold.

The use of simple calix[4]arenes for chemical conversion of NO2/N2O4 gases is demonstrated in solution and in the solid state. Upon reacting with these gases, calixarenes 1 encapsulate nitrosonium (NO+) cations within their cavities with the formation of stable calixarene-NO+ complexes 2. These complexes act as encapsulated nitrosating reagents; cavity effects control their reactivity and selectivity. Complexes 2 were effectively used for nitrosation of secondary amides 5, including chiral derivatives. Unique size-shape selectivity was observed, allowing for exclusive nitrosation of less crowded N-Me amides 5 a-e (up to 95 % yields). Bulkier N-Alk (Alk>Me) substrates 5 did not react due to the hindered approach to the encapsulated NO+ reagents. Robust, silica gel based calixarene material 3 was prepared, which reversibly traps NO2/N2O4 with the formation of NO+-storing silica gel 4. With material 4, similar size-shape selectivity was observed for nitrosation. The N-Me-N-nitroso derivatives 6 d,e were obtained with approximately 30 % yields, while bulkier amides were nitrosated with much lower yields (

Supramolecular approaches to visual detection, storage, conversion and use of NO X gases are described that deploy calixarenes and their derivatives, such as hemicarcerands and synthetic nanotubes. Polymer-supported calixarene materials for NO X are also introduced. Chemical fixation of NO2/N2O4 is demonstrated through the transformation into calixarene–NO+ complexes and their use as nitrosonium storing and releasing reagents for organic synthesis. These findings highlight perspectives of supramolecular chemistry and molecular recognition in sensing and fixation of environmentally important gases.

The use of simple calix[4]arenes 1a,b for NO2/N2O4 sensing and conversion is demonstrated, both in solution and in the solid state. Upon reacting with these gases, compounds 1a,b encapsulate reactive NO+ cations within their cavities with the formation of deeply colored (λmax∼570 nm) charge-transfer complexes 2a,b . Further functionalization of the calix[4]arene platform is reported for attachment to solid supports. Polymer-supported calixarene material 3 was prepared, which reversibly traps NO2/N2O4 with the formation of nitrosonium storing polymer 4 . Material 4 was effectively used for nitrosation of amides.

An approach toward visual detection and chemical utilization of NO(2)/N(2)O(4) is proposed, which employs simple calix[4]arenes. Exposure of tetra-O-alkylated calix[4]arenes 1 and 2, possessing either a cone or a 1,3-alternate conformation, to NO(2)/N(2)O(4), both in chloroform solution and in the solid state, results in deeply colored calixarene-nitrosonium (NO(+)) complexes. In the presence of a Lewis acid, such as SnCl(4), stable calixarene-NO(+) complexes 7 and 8 were isolated in a quantitative yield and characterized by UV-vis, FTIR, high-resolution (1)H NMR spectroscopy and elemental analysis. NO(+) is found encapsulated within the calixarene cavity, and stable charge-transfer complexes result with K(ass)10(6) M(-1) (CDCl(3)). The NO(+) encapsulation was also demonstrated in titration experiments with calixarenes 1, 2, and 5 and commercially available NO(+)SbF(6)(-) salt in chloroform. The complexation process is reversible, and the complexes dissociate upon addition of water and alcohol, recovering the parent calixarenes. Attachment of functionalized calix[4]arenes to silica gel was demonstrated, which afforded a solid material 15 capable of visual detection and entrapment of NO(2)/N(2)O(4). Calixarene-NO(+) complexes can be utilized for the NO(+) transfer processes and nitrosation reactions. The NO(+) guest transfer between two calixarene containers 2 and 5 was achieved and studied by UV-vis and (1)H NMR spectroscopy. Chemical fixation of NO(2)/N(2)O(4) was demonstrated through their quantitative transformation into the calixarene-NO(+) complex and its use as a nitrosonium transfer agent in the synthesis of N-nitrosoamides. These results may lead toward novel nitrogen oxides storing materials.

Molecular chaperone heat shock protein 90 (Hsp90) is an important target in cancer and neurodegenerative diseases, and has rapidly become the focus of several drug discovery efforts. Among small molecule Hsp90 inhibitors with clinical applicability are derivatives of 8-arylmethyl-9H-purin-6-amine class. Here we report the use of microwave-assisted chemistry for the successful one-pot delivery of 8-arylmethyl-9H-purin-6-amines. We discuss the applicability as well as the limitations of this method towards the creation of a large chemical diversity in the 8-arylmethyl-9H-purin-6-amine series.

The 70 kDa heat shock proteins (Hsp70) are molecular chaperones that assist in folding of newly synthesized polypeptides, refolding or denaturation of misfolded proteins, and translocation of proteins across biological membranes. In addition, Hsp70 play regulatory roles in signal transduction, cell cycle, and apoptosis. Here, we present a novel assay platform based on fluorescence polarization that is suitable for investigating the yet elusive molecular mechanics of human Hsp70 allosteric regulation.

The heat-shock protein 90 (Hsp90), an important target in cancer and other diseases, has become recently the focus of several drug discovery and development efforts. The initially identified natural-product inhibitors of Hsp90, such as geldanamycin, played a major role in elucidating its biological function and in determining its clinical relevance. Upcoming synthetic inhibitors, such as the purine-scaffold class, furthered our understanding on Hsp90 in cancer and neurodegenerative diseases and delivered what are promised to be clinical candidates with favorable pharmacologic profiles. This review intends to inform the reader on efforts ranging from the discovery of purine-scaffold Hsp90 inhibitors to their clinical translation as well as on their use as chemical tools to dissect the roles of Hsp90 in pathogenic systems.

Molecular chaperone heat shock protein 90 (Hsp90) is an important target in cancer and neurodegenerative diseases, and has rapidly become the focus of several drug discovery efforts. Among small molecule Hsp90 inhibitors with clinical applicability are derivatives of 8-arylmethyl-9H-purin-6-amine class. Here we report the use of microwave-assisted chemistry for the successful one-pot delivery of 8-arylmethyl-9H-purin-6-amines. We discuss the applicability as well as the limitations of this method towards the creation of a large chemical diversity in the 8-arylmethyl-9H-purin-6-amine series.

PU-H71 is a purine-scaffold Hsp90 inhibitor currently undergoing late stage preclinical evaluation for the treatment of cancer. In this investigation, we present a simple method for the synthesis of d6-labeled PU-H71 for use as an internal standard to accurately quantitate the drug in biological matrices based on an LC-MS-MS method. PU-H71-d6 was synthesized in five steps using readily available 1,3-dibromopropane-d6 and is an important compound for the advancement of our clinical program. Copyright © 2009 John Wiley & Sons, Ltd.

A novel high-speed optical switch based on organic coumarin waveguide is presented. The device principle is based on influencing the bend loss in a waveguide by changing the refractive index contrast defining the guide. A low power femtosecond laser is used to excite the cladding of the curved waveguide. The beam propagation method is employed in the numerical simulation. The designed optical switch has a large extinction ratio and rapid response time (about 1.2ps).

The heat shock protein 90 (Hsp90) has a critical role in malignant transformation. Whereas its ability to maintain the functional conformations of mutant and aberrant oncoproteins is established, a transformation-specific regulation of the antiapoptotic phenotype by Hsp90 is poorly understood. By using selective compounds, we have discovered that small-cell lung carcinoma is a distinctive cellular system in which apoptosis is mainly regulated by Hsp90. Unlike the well-characterized antiapoptotic chaperone Hsp70, Hsp90 is not a general inhibitor of apoptosis, but it assumes this role in systems such as small-cell lung carcinoma, in which apoptosis is uniquely dependent on and effected through the intrinsic pathway, without involvement of caspase elements upstream of mitochondria or alternate pathways that are not apoptosome-channeled. These results provide important evidence for a transformation-specific interplay between chaperones in regulating apoptosis in malignant cells.

Background: Heat shock protein 70 family members play an important role in cancer. They are up-regulated in wide variety of tumors and the increased Hsp70 protein expression correlates with metastases, resistance to treatment and poor prognosis. Multiple mechanisms explain cancer cells dependence on Hsp70, such as inhibition of apoptosis by Hsp70, induction of autophagy and control of stability of onco-proteins. These Hsp70 activities are mediated in cancer by its ability to chaperone and interact with a large number of proteins in a cell-specific, context dependent manner. Hypothesis: Reagents that enable the capture of tumor-specific Hsp70 complexes facilitate the identification of context-dependent Hsp70 interactomes. Results: Our laboratory recently reported the identification of a novel allosteric site located in the nucleotide binding domain of Hsp70 (Chem Biol 2013). It has also reported the discovery of ligands that bind to the allosteric pocket of Hsp70, inhibit its function in cancer cells and result in anti-cancer activity (J Med Chem 2013). Structure-activity relationship studies in this ligand series gave insights on the attachment of specific linkers for the design of Hsp70-related chemical tools. Here we present the design of Hsp70-directed reagents and use biochemical and cell-based methods to validate Hsp70-directed affinity purification probes. We demonstrate that these tools lock Hsp70 in complex with onco-client proteins and effectively isolate Hsp70 complexes for identification through biochemical techniques. Using these tools we provide proof-of-concept analyses that glimpse into the complex roles played by Hsp70 in maintaining a multitude of cell-specific malignancy-driving proteins. Significance: The knowledge derived from the use of such reagents will be extremely valuable not only to understand tumor-specific roles of Hsp70 and associated mechanisms but also to develop rational strategies for the clinical implementation of these agents to cancer treatment. They may also provide clues on the altered functional proteome in individual tumors, a quest yet elusive by today's proteomics methods. Citation Format: Anna A. Rodina, Tony Taldone, Yanlong Kang, Pallav Patel, John Koren, Pengrong Yan, Erica DaGama Gomes, Chenghua Yang, Maulik Patel, Liza Shrestha, Stefan Ochiana, Ronnie Maharaj, Alexander Gozman, Marc Cox, Hediye Erdjument-Bromage, Ronald Hendrickson, Leandro Cerchietti, Ari Melnick, Monica Guzman, Gabriela Chiosis. Development of chemical tools to study the endogenous Hsp70 interactome in malignant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2015-1733

As molecularly targeted agents assume a more prominent role in anticancer therapy there is a growing need to determine in a noninvasive manner whether the target is being engaged and to what extent such drug-target binding results in desirable effects. We address this need in the context of Hsp90, a target of significant value and one in critical need for such assessment tools, by combining a novel chemical tool selective for tumor Hsp90 with PET imaging and mathematical modeling. The chemical tool is [124I]-PU-H71, the iodine-124 radiolabeled analog of the potent Hsp90 inhibitor PU-H71, which can be administered in tracer quantities for PET imaging. The resulting diagnostic, PU-PET, has been optimized and validated preclinically in mouse models of cancer and then translated to the clinic. The exquisite design of this assay is based on three essential concepts as it relates to the target (Hsp90) as well as to the PET tracer (124I-PU-H71). First, the target is “oncogenic” Hsp90 and has been shown by numerous biochemical and pharmacokinetic studies to have a strong affinity for inhibitors and a very low koff resulting in selective and prolonged retention in tumor. Secondly, the tracer incorporates a 124I in place of the naturally occurring 127I in the structure of PU-H71 and therefore there is no change in the chemical structure. This feature in a PET tracer intended as a companion diagnostic is unprecedented and ensures that the PK properties are identical to the therapeutic agent (PU-H71). Finally, the radionuclide 124I has a four-day half-life and thus is well-suited to monitor the extended tumor retention profile observed for Hsp90 inhibitors. We here demonstrate that this PET assay informs on Hsp90 targeting in individual tumors in real time and provides accurate tumor drug concentrations for at least four chemically distinct Hsp90 drugs. In contrast, we find that plasma pharmacokinetics is not predictive of intratumor parameters and therefore provides limited value in estimating target engagement. Using PU-PET we demonstrate that at least one Hsp90 inhibitor exhibits tumor targeting and retention in humans, delivering and retaining therapeutic, micromolar, concentrations at safe doses. PU-PET is currently being evaluated in Phase 0/1 (NCT01269593) clinical trials as a noninvasive companion diagnostic to determine intratumoral concentration as well as to identify those patients who would best benefit from Hsp90 inhibitor therapy. This diagnostic assay is intended to be incorporated into future Phase 2 clinical trials in order to preselect those patients who would most likely benefit from Hsp90 inhibitor treatment. Citation Format: Tony Taldone, Nagavarakishore Pillarsetty, Mark PS Dunphy, John F. Gerecitano, Eloisi Caldas-Lopes, Brad Beattie, Radu I. Peter, Yanlong Kang, Anna Rodina, Pengrong Yan, Erica M. DaGama Gomes, Alexander Bolaender, Christina Pressl, Blesida Punzalan, Anson Ku, Thomas Ku, Smit Shah, Mohammad Uddin, Mei H. Chen, Elmer Santos, Jacek Koziorowski, Adriana Corben, Shanu Modi, Komal Jhaveri, Oscar Lin, Efsevia Vakiani, Yelena Janjigian, Pat Zanzonico, Clifford Hudis, Steven M. Larson, Jason S. Lewis, Gabriela Chiosis. Development of a noninvasive assay to determine drug concentration in tumor during hsp90 inhibitor therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5444. doi:10.1158/1538-7445.AM2015-5444

The synthesis of a red-shifted cy3B-GM ligand and its evaluation as a fluorescence polarization probe for Hsp90 is presented.

Reaction of NO2 with simple calix[4]arenes in chloroform in the presence of a Lewis acid rapidly results in intense coloration caused by the encapsulation of nitrosonium cation.

The 70 kDa heat shock protein (Hsp70) plays an important role in cancer and its pharmacological modulation with small molecules may represent a useful therapeutic approach. The discovery of such agents has been hampered by the lack of a full length crystal structure of human Hsp70. We constructed a homology model of the human Hsp70, which we investigated for potential druggable sites using SiteMap tool. An allosteric site in the nucleotide binding domain was identified and used to design potent Hsp70 modulators. Analysis of the Glide docking resulted in an understanding of important interactions between designed ligands, such as YK5, and the Hsp70 protein. When tested in breast cancer cells, YK5 led to depletion of onco-proteins, induced apoptosis and inhibited growth. YK5 is to our knowledge the first rationally designed small molecule inhibitor of Hsp70, and thus, represents a novel chemical tool to investigate its potential in cancer and other diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3895. doi:1538-7445.AM2012-3895

Abstract 2493 Prognosis for acute myelogenous leukemia (AML) with traditional chemotherapeutic agents remains poor, most patients relapse and die of their disease. Studies have shown that AML can be originated and maintained by leukemia stem cells (LSCs) and that a high percentage of phenotypically-defined LSCs correlate to especially poor prognosis. Thus, novel therapies that can eliminate LSCs are needed. Upregulation of HSP70 has been described in human cancer cells and is often associated with chemoresistance and poor prognosis. Therefore, HSP70 represents a novel target for cancer therapy. Due to this, we tested a novel inhibitor of tumor-HSP70, YK5, and found that it can induce potent cell death in AML cells including progenitor and stem cell populations with minimal effects in normal hematopoietic cells. Fifteen primary AML patient samples were treated with YK5 in varying concentrations for 48 hours to determine the LD50 of YK5. The viability of these samples was determined by multiparameter flow cytometry using cell surface markers to distinguish, lymphocytes, blasts, progenitor and stem cell populations in conjunction with Annexin V and 7AAD. Mean LD50 for all samples tested was 4.5μM (CI 95% 2.239–6.853). Three of these samples had very low LD50s below 2.5μM and only one sample demonstrated relative resistance with a LD50 that was greater than 15μM. Notably, progenitor and stem cell populations were also targeted while normal remaining lymphocytes were spared. Furthermore, we found that LSCs in a subset of patient samples treated with YK5 exhibited higher sensitivity to the compound when compared to blast cells. Importantly, treatment of primary AML samples with 5μM YK5 resulted in a 63% decrease in colony forming ability when compared to untreated control (N=5), suggesting that YK5 can impair malignant progenitors. In contrast, normal CD34+ cord blood cells after treatment with 5μM YK5 for 48hrs exhibited a viability of 57% (N=3). Xenotransplant assays are currently ongoing to determine the ability of YK5 to inhibit LSC function. Treatment with YK5 resulted in a decrease in phosphorylation of STAT5, determined by flow cytometry. Taken together, HSP70 inhibition is a promising potential treatment for AML and has the potential to eradicate leukemic stem and progenitor cells while sparing normal hematopoietic cells. Thus, targeting HSP70 in AML appears to hold great promise. *JFR and KKS contributed equally to this project Disclosures: Roboz: EpiCept: Consultancy; ChemGenex: Consultancy; Celgene: Consultancy; Boehringer Ingelheim: Consultancy.

Background. The cancerous phenotype evolves through accumulation of a large repertoire of elements that allow for increased proliferative potential, self-sufficiency in growth signals and resistance to apoptotic and antiproliferative signals. While much is now gained in understanding the many factors that lead to a malignant phenotype, little remains known on how cancer cells are able to accumulate and to survive with such complexity and high load of aberrant processes. Study design. Here we use the novel dual Hsp70 and Hsc70 inhibitor, YK5, and its biotinylated derivative, YK55, to investigate the interactome of cytosolic Hsp70s. Results. We show that heat shock protein 70 family members (Hsp70s) keep active conformations of key oncoproteins and inhibit the function of tumor suppressors. Hsp70s also buffer levels of activated molecules by maintaining them in a conformation that alters their dephosphorylation by phosphatases. Pharmacologic inhibition of Hsp70s by YK5 impairs oncoprotein stability and function, restores tumor suppressor activity, affects major cancer hallmarks and is selectively lethal to cancer cells. We show that enhanced affinity of YK5 for cancer cell-Hsp70s species is an essential mechanism for providing selective tumor lethality. Conclusion. By demonstrating that pharmacologic dual inhibitors of Hsp70 and Hsc70, such as YK5, confer specific and broad tumor lethality, and interact selectively with tumor Hsp70s species, this work proposes Hsp70s inhibitors as potential anti-cancer therapeutics with a large spectrum of activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2579. doi:10.1158/1538-7445.AM2011-2579

A Ru(II) porphyrin rapidly reacts with NO2/N2O4 with the formation of a stable Ru(II) nitrosyl nitrato complex.