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• W2080409112 abstract "The molecular chaperone and cytoprotective activities of the Hsp70 and Hsp40 chaperones represent therapeutic targets for human diseases such as cancer and those that arise from defects in protein folding; however, very few Hsp70 and no Hsp40 modulators have been described. Using an assay for ATP hydrolysis, we identified and screened small molecules with structural similarity to 15-deoxyspergualin and NSC 630668-R/1 for their effects on endogenous and Hsp40-stimulated Hsp70 ATPase activity. Several of these compounds modulated Hsp70 ATPase activity, consistent with the action of NSC 630668-R/1 observed previously (Fewell, S. W., Day, B. W., and Brodsky, J. L. (2001) J. Biol. Chem. 276, 910–914). In contrast, three compounds inhibited the ability of Hsp40 to stimulate Hsp70 ATPase activity but did not affect the endogenous activity of Hsp70. Two of these agents also compromised the Hsp70/Hsp40-mediated post-translational translocation of a secreted pre-protein in vitro. Together, these data indicate the potential for continued screening of small molecule Hsp70 effectors and that specific modulators of Hsp70-Hsp40 interaction can be obtained, potentially for future therapeutic use. The molecular chaperone and cytoprotective activities of the Hsp70 and Hsp40 chaperones represent therapeutic targets for human diseases such as cancer and those that arise from defects in protein folding; however, very few Hsp70 and no Hsp40 modulators have been described. Using an assay for ATP hydrolysis, we identified and screened small molecules with structural similarity to 15-deoxyspergualin and NSC 630668-R/1 for their effects on endogenous and Hsp40-stimulated Hsp70 ATPase activity. Several of these compounds modulated Hsp70 ATPase activity, consistent with the action of NSC 630668-R/1 observed previously (Fewell, S. W., Day, B. W., and Brodsky, J. L. (2001) J. Biol. Chem. 276, 910–914). In contrast, three compounds inhibited the ability of Hsp40 to stimulate Hsp70 ATPase activity but did not affect the endogenous activity of Hsp70. Two of these agents also compromised the Hsp70/Hsp40-mediated post-translational translocation of a secreted pre-protein in vitro. Together, these data indicate the potential for continued screening of small molecule Hsp70 effectors and that specific modulators of Hsp70-Hsp40 interaction can be obtained, potentially for future therapeutic use. The constitutively expressed and stress-inducible 70-kDa heat shock proteins Hsc70 and Hsp70, respectively, are ubiquitous molecular chaperones that bind and release polypeptides in an ATP-dependent cycle. These chaperones contain three interdependent domains, which are a highly conserved 44-kDa N-terminal ATPase domain, an 18-kDa peptide binding domain, and a 10-kDa C-terminal helical lid domain (1Chappell T.G. Konforti B.B. Schmid S.L. Rothman J.E. J. Biol. Chem. 1987; 262: 746-751Abstract Full Text PDF PubMed Google Scholar, 2Flaherty K.M. Deluca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (833) Google Scholar, 3Morshauser R.C. Wang H. Flynn G.C. Zuiderweg E.R. Biochemistry. 1995; 34: 6261-6266Crossref PubMed Scopus (94) Google Scholar, 4Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar, 5Wang T.F. Chang J.H. Wang C. J. Biol. Chem. 1993; 268: 26049-26051Abstract Full Text PDF PubMed Google Scholar). The lid domain gates the polypeptide binding pocket in an ATP-dependent fashion such that when ATP is bound in the N-terminal domain, the peptide binding channel is exposed and Hsp70s exhibit fast on and off rates for substrate binding. Transient interactions with peptide can stimulate ATP hydrolysis, triggering a conformational change in the chaperone (6Schmid D. Baici Gehring H. Christen P. Science. 1994; 263: 971-973Crossref PubMed Scopus (422) Google Scholar, 7McCarty J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (349) Google Scholar). This increases the affinity of Hsp70s for peptides by closing the lid domain on the peptide binding pocket and trapping bound substrates (8Misselwitz B. Staeck O. Rapoport T.A. Mol. Cell. 1998; 2: 593-603Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Ultimately, peptide substrates are released concomitant with the exchange of ADP for ATP. Hsp70 function is regulated by and often dependent upon Hsp40 co-chaperones, which are defined by their homology to the DnaJ chaperone in Escherichia coli. Hsp40s stimulate the ATPase activity of Hsp70s and, thus, stabilize the Hsp70-peptide complex. An ∼70-amino acid “J domain” defines the Hsp40 family and interacts with the ATPase domain of Hsp70 (for review, see Ref. 9Cheetham M.E. Caplan A.J. Cell Stress Chaperones. 1998; 3: 28-36Crossref PubMed Scopus (495) Google Scholar). Other regions in Hsp40 chaperones might interact with Hsp70 near its peptide binding domain (10Suh W.-C. Burkholder W.F. Lu C.Z. Zhao X. Gottesman M.E. Gross C.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15223-15228Crossref PubMed Scopus (228) Google Scholar, 11Greene L.E. Zinner R. Naficy S. Eisenberg E. J. Biol. Chem. 1995; 270: 2967-2973Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 12Gassler C.S. Buchberger A. Laufen T. Mayer M.P. Schroder H. Valencia A. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15229-15234Crossref PubMed Scopus (151) Google Scholar). Hsp40s can orchestrate substrate delivery to the peptide binding domain of Hsp70 (13Wickner S. Hoskins J. McKenney K. Nature. 1991; 350: 165-167Crossref PubMed Scopus (159) Google Scholar, 14Langer T. Lu C. Echols H. Flanagan J. Hayer M.K. Hartl F.U. Nature. 1992; 356: 683-689Crossref PubMed Scopus (793) Google Scholar, 15Szabo A. Langer T. Schroder H. Flanagan J. Bukau B. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10345-10349Crossref PubMed Scopus (444) Google Scholar, 16Laufen T. Mayer M.P. Beisel C. Klostermeier D. Mogk A. Reinstein J. Bukau B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5452-5457Crossref PubMed Scopus (475) Google Scholar, 17Han W. Christen P. J. Biol. Chem. 2003; 278: 19038-19043Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Gamer J. Multhaup G. Tomoyasu T. McCarty J.S. Rudiger S. Schonfeld H.J. Schirra C. Bujard H. Bukau B. EMBO J. 1996; 15: 607-617Crossref PubMed Scopus (241) Google Scholar) and have been shown to expand the diversity of Hsp70s substrates (8Misselwitz B. Staeck O. Rapoport T.A. Mol. Cell. 1998; 2: 593-603Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Because not all Hsp40 J domains are interchangeable (19Schlenstedt G. Harris S. Risse B. Lill R. Silver P.A. J. Cell Biol. 1995; 129: 979-988Crossref PubMed Scopus (134) Google Scholar, 20McClellan A.J. Endres J.B. Vogel J.P. Palazzi D. Rose M.D. Brodsky J.L. Mol. Biol. Cell. 1998; 9: 3533-3545Crossref PubMed Scopus (73) Google Scholar, 21Sullivan C.S. Tremblay J.D. Fewell S.W. Lewis J.A. Brodsky J.L. Pipas J.M. Mol. Cell. Biol. 2000; 20: 5749-5757Crossref PubMed Scopus (74) Google Scholar, 22Cyr D.M. Lu X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar), Hsp40s may also dictate the specificity of Hsp70 function. Because they are involved in protein folding and quality control, Hsp70 and Hsp40 co-chaperone functions represent a therapeutic target for human diseases caused by protein folding defects, such as cystic fibrosis (23Brodsky J.L. Am. J. Physiol. Lung Cell. Mol. Physiol. 2001; 281: 39-42Crossref PubMed Google Scholar, 24Gelman M.S. Kopito R.R. J. Clin. Investig. 2002; 110: 1591-1597Crossref PubMed Scopus (81) Google Scholar). The folding and subsequent trafficking beyond the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; BiP, IgG heavy chain-binding protein; CFTR, cystic fibrosis transmembrane conductance regulator; DSG, 15-deoxyspergualin; R/1, NSC 630368-R/1; SV40, simian virus 40; TAg, simian virus 40 large T antigen; ppαF, prepro-α-factor. of an unstable, mutated form of the cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR) is enhanced in cells exposed to chemical chaperones (e.g. glycerol, trimethylamine oxide (TMAO), inositol, taurine, or sorbitol (25Brown C.R. Hong-Brown L.Q. Biwersi J. Verkman A.S. Welch W.J. Cell Stress Chaperones. 1996; 1: 117-125Crossref PubMed Scopus (362) Google Scholar, 26Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-638Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar, 27Howard M. Fischer H. Roux J. Santos B.C. Gullans S.R. Yancey P.H. Welch W.J. J. Biol. Chem. 2003; 278: 35159-35167Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), or compounds that modulate Hsp70 activity or levels (e.g. 15-deoxyspergualin or phenylbutyrate (28Jiang C. Fang S.L. Xiao Y.F. O'Connor S.P. Nadler S.G. Lee D.W. Jefferson D.M. Kaplan J.M. Smith A.E. Cheng S.H. Am. J. Physiol. 1998; 275: C171-C178Crossref PubMed Google Scholar, 29Rubenstein R.C. Zeitlin P.L. Am. J. Physiol. Cell Physiol. 2000; 278: C259-C267Crossref PubMed Google Scholar)). Combined with modulators that enhance the activity of ΔF508-CFTR at the plasma membrane (30Yang H. Shelat A.A. Guy R.K. Gopinath V.S. Ma T. Du K. Lukacs G.L. Taddei A. Folli C. Pedemonte N. Galietta L.J. Verkman A.S. J. Biol. Chem. 2003; 278: 35079-35085Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), it is hoped that analogous compounds might ultimately ameliorate the adverse phenotypes of this disease. Hsp70 and Hsp40 also contribute to tumorigenesis (31Jolly C. Morimoto R.I. J. Natl. Cancer Inst. 2000; 92: 1564-1572Crossref PubMed Scopus (879) Google Scholar, 32Beere H.M. Green D.R. Trends Cell Biol. 2001; 11: 6-10Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Several unique tumor types exhibit elevated expression of these chaperones (33Ciocca D.R. Clark G.M. Tandon A.K. Fuqua S.A. Welch W.J. McGuire W.L. J. Natl. Cancer Inst. 1993; 85: 570-574Crossref PubMed Scopus (323) Google Scholar, 34Kaur J. Ralhan R. Int. J. Cancer. 1995; 63: 774-779Crossref PubMed Scopus (57) Google Scholar, 35Ralhan R. Kaur J. Clin. Cancer Res. 1995; 1: 1217-1222PubMed Google Scholar, 36Santarosa M. Favaro D. Quaia M. Galligioni E. Eur. J. Cancer. 1997; 33: 873-877Abstract Full Text PDF PubMed Scopus (104) Google Scholar), and overexpression of Hsp70 alone can lead to cellular transformation (37Volloch V.Z. Sherman M.Y. Oncogene. 1999; 18: 3648-3651Crossref PubMed Scopus (105) Google Scholar) and tumorigenesis (38Seo J.S. Park Y.M. Kim J.I. Shim E.H. Kim C.W. Jang J.J. Kim S.H. Lee W.H. Biochem. Biophys. Res. Commun. 1996; 218: 582-587Crossref PubMed Scopus (97) Google Scholar). Consistent with these observations, lowering the level of Hsp70 using antisense technology inhibits the proliferation of breast cancer cells by inducing apoptosis (39Nylandsted J. Rohde M. Brand K. Bastholm L. Elling F. Jaattela M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7871-7876Crossref PubMed Scopus (357) Google Scholar). In addition, some viral oncogenes recruit cellular Hsp70 to inactivate growth control checkpoints (40Sullivan C.S. Pipas J.M. Virology. 2001; 287: 1-8Crossref PubMed Scopus (74) Google Scholar). The large T antigen (TAg) of the DNA tumor virus, simian virus 40 (SV40), contains a J domain that stimulates Hsp70 to rearrange multiprotein complexes involved in cell cycle regulation (41Sullivan C.S. Cantalupo P. Pipas J.M. Mol. Cell. Biol. 2000; 20: 6233-6243Crossref PubMed Scopus (103) Google Scholar). Thus, modulators of Hsp70 and Hsp40 activity might also serve as anti-cancer drugs. Three compounds that modulate the ATPase activity of Hsp70 have been described. One compound, 15-deoxyspergualin (DSG), binds Hsp70 (KD = 4 μm) and stimulates its steady-state ATPase activity by 20–40% (42Nadler S.G. Eversole A.C. Tepper M.A. Cleaveland J.S. Ther. Drug Monit. 1995; 17: 700-703Crossref PubMed Scopus (48) Google Scholar, 43Brodsky J.L. Biochem. Pharmacol. 1999; 57: 877-880Crossref PubMed Scopus (46) Google Scholar). DSG is currently being used in clinical trials to combat the rejection of transplanted kidneys (44Tanabe K. Tokumoto T. Ishikawa N. Shimizu T. Okuda H. Ito S. Shimmura H. Inui M. Harano M. Ohtsubo S. Manu M. Shiroyanagi Y. Yagisawa T. Fuchinoue S. Toma H. Transplant. Proc. 2000; 32: 1745-1746Crossref PubMed Scopus (8) Google Scholar) and has been shown in vitro to modestly facilitate the trafficking of ΔF508-CFTR to the plasma membrane (28Jiang C. Fang S.L. Xiao Y.F. O'Connor S.P. Nadler S.G. Lee D.W. Jefferson D.M. Kaplan J.M. Smith A.E. Cheng S.H. Am. J. Physiol. 1998; 275: C171-C178Crossref PubMed Google Scholar). A second compound that bears structural similarity to DSG was identified, NSC-630668-R/1 (designated R/1), which inhibits the endogenous and Hsp40-stimulated ATPase activity of Hsp70 by 48 and 51% (45Fewell S.W. Day B.W. Brodsky J.L. J. Biol. Chem. 2001; 276: 910-914Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). R/1 also prevents the translocation of a pre-protein into yeast ER-derived vesicles, a process that requires cytosolic and lumenal Hsp70 and Hsp40 co-chaperones (45Fewell S.W. Day B.W. Brodsky J.L. J. Biol. Chem. 2001; 276: 910-914Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Finally, 3′-sulfogalactolipids bind the ATPase domain of Hsp70 (46Mamelak D. Lingwood C. J. Biol. Chem. 2001; 276: 449-456Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and inhibit the endogenous and Hsp40-stimulated steady-state ATPase cycle (47Whetstone H. Lingwood C. Biochemistry. 2003; 42: 1611-1617Crossref PubMed Scopus (44) Google Scholar). Given the importance of Hsp70-Hsp40 function on cellular physiology, it is imperative that additional modulators with unique properties be discovered. To this end we identified and then biochemically screened structural analogs of DSG and R/1 for their effects on the endogenous and Hsp40-stimulated ATPase activity of a yeast Hsp70. We employed a sensitive assay to measure the rate of Hsp70 ATP hydrolysis in the presence or absence of the different compounds and for the first time obtained unique classes of chaperone modulators. Identification and Synthesis of DSG/NSC 630668-R/1 Analogs— After computational comparison of the DSG and R/1 structures (Fig. 1 (45Fewell S.W. Day B.W. Brodsky J.L. J. Biol. Chem. 2001; 276: 910-914Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar)), analogs were identified by sub-structural analogy searches against libraries of compounds resident in the Developmental Therapeutics Program at the National Cancer Institute and in the University of Pittsburgh Center for Chemical Methodologies and Library Development. The following compounds were obtained from the Developmental Therapeutics Program: NSC 624392, NSC 624393, NSC 624903, NSC 624904, NSC 624905, NSC 624906, NSC 624907, NSC 624908, NSC 625194, NSC 625195, NSC 625512, NSC 625513, NSC 632006, and NSC 655302. The synthesis of SC-ααδ9 was previously described (48Wipf P. Cunningham A. Rice R.L. Lazo J.S. Bioorg. Med. Chem. 1997; 5: 165-177Crossref PubMed Scopus (51) Google Scholar). The ML2 and MAL3 series of compounds were prepared by Ugi and Biginelli reactions. The one-pot cyclocondensation of β-ketoesters 1, aromatic aldehydes 2, and urea 3 was performed in tetrahydrofuran at room temperature in the presence of catalytic amounts of HCl (49Wipf P. Cunningham A. Tetrahedron Lett. 1995; 36: 7819-7822Crossref Scopus (314) Google Scholar) or by heating in N,N-dimethylformamide to give heterocycle 4 (Fig. 2A). Although yields depended strongly on the substitution pattern and ranged from 33 to 84% for 4a-4e, purities determined by liquid chromatography-mass spectrometry analysis (TIC MSD) were excellent and uniformly exceeded 90%. Subsequently, the N-1-substitued Biginelli dihydropyrimidinones 4a-4e were subjected to a second multicomponent reaction, the Ugi condensation (50Domling A. Ugi I.I. Angew. Chem. Int. Ed. Engl. 2000; 39: 3168-3210Crossref PubMed Google Scholar, 51Studer A. Jeger P. Wipf P. Curran D.P. J. Org. Chem. 1997; 62: 2917-2924Crossref PubMed Scopus (278) Google Scholar). Although standard thermal conditions provided disappointing conversions in this reaction, microwave conditions using the CEM Discover™ Microwave reactor were more successful. A stirred solution of 4a-e and amine in methanol was treated with aldehyde and n-butyl or isocyanide at room temperature. The mixture was then heated twice at 70 °C for 20–30 min in the microwave reactor. The MAL3 Ugi-Biginelli products were purified by column chromatography and analyzed by liquid chromatography-mass spectrometry. Finally, samples were dissolved in dimethyl sulfoxide (Me2SO) and stored at 4 °C. Structure-Activity Analysis—A computational conformational analysis was performed on R/1 using the Boltzman jump stochastic search method in Cerius2 (v.4.5 Accelrys, Inc.). Because R/1 is a very flexible molecule with many rotatable bonds, we decomposed the molecular model of R/1 into fragments containing 4–7 rotatable bonds and assumed that the minimum energy structure obtained for each was a good local approximation of the structure in the corresponding region of the whole molecule. Conformational energies were computed using Merck Molecular Force Field (MMFF94) with no cutoffs. The resulting minimum energy conformer obtained for R/1 suggested that the presence of two carbamic ester moieties equally distributed between the two uracil rings (separated by six methylene groups) allows for the possibility of hydrogen bonding between the carbamic ester moieties and the hydrogen donors/acceptors from the uracil substituents and also hydrophobic interactions between the hydrocarbon chains that separate the two carbamic ester moieties from each other and from the uracil groups. These hydrophobic interactions may promote the packing of the hydrocarbon chain into a low energy conformer. Molecular mechanics-minimized models of the 31 test compounds were aligned with the hypothetical structure of R/1 described above using Cerius2 (for example, see Fig. 2B). These aligned structures and the activity data were then used to define descriptors for the dataset, including steric, electrostatic and hydrogen donor/acceptor fields along with dipole moment and ClogP descriptors. ATPase Measurements—Yeast Hsp70 (Ssa1p), Ydj1p, and SV40 large TAg were purified as described previously (22Cyr D.M. Lu X. Douglas M.G. J. Biol. Chem. 1992; 267: 20927-20931Abstract Full Text PDF PubMed Google Scholar, 52Caplan A.J. Tsai J. Casey P.J. Douglas M.G. J. Biol. Chem. 1992; 267: 18890-18895Abstract Full Text PDF PubMed Google Scholar, 53Cantalupo P. Saenz-Robles M.T. Pipas J.M. Methods Enzymol. 1999; 306: 297-307Crossref PubMed Scopus (12) Google Scholar, 54McClellan A.J. Brodsky J.L. Genetics. 2000; 156: 501-512Crossref PubMed Google Scholar). Assays that approximate single turnover measurements of endogenous Hsp70 ATPase activity were performed at 30 °C on pre-formed [α32P]ATP-Hsp70 complexes according to published methods (21Sullivan C.S. Tremblay J.D. Fewell S.W. Lewis J.A. Brodsky J.L. Pipas J.M. Mol. Cell. Biol. 2000; 20: 5749-5757Crossref PubMed Scopus (74) Google Scholar, 45Fewell S.W. Day B.W. Brodsky J.L. J. Biol. Chem. 2001; 276: 910-914Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 55Ziegelhoffer T. Lopez-Buesa P. Craig E.A. J. Biol. Chem. 1995; 270: 10412-10419Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 56Miao B. Davis J.E. Craig E.A. J. Mol. Biol. 1997; 265: 541-552Crossref PubMed Scopus (82) Google Scholar). Briefly, 25 μg of Hsp70 was incubated with 100 μCi of [α32P]ATP (PerkinElmer Life Sciences; 3000 Ci/mmol) on ice for 30 min in Complex buffer (100 mm Hepes-KOH (pH 7.5), 300 mm KCl, 80 mm magnesium acetate) and 25 μm ATP in a final volume of 100 μl (Step 1, Fig. 3A). After this incubation, the [α32P]ATP-Hsp70 complex was purified from free [α32P]ATP on a NICK G-50 column (Amersham Biosciences) at 4 °C (Step 2, Fig. 3A). Glycerol was added to a final concentration of 10%, and 25-μg aliquots (∼0.6 μm Hsp70) were frozen in liquid nitrogen and stored at -80 °C no longer than 3 weeks. To assay ATP hydrolysis, individual aliquots were rapidly thawed and added to an equal volume of Complex buffer containing test compounds or Me2SO pre-equilibrated to 30 °C (Step 3, Fig. 3A). The final concentration of Hsp70 was ∼0.2–0.3 μm in these reactions. At the specified time points, 6-μl aliquots were removed and added to 2 μl of stop solution (2 m LiCl, 4 m formic acid, 36 mm ATP) on ice to quench ATP hydrolysis. Aliquots of each reaction time point were spotted onto thin layer chromatography (TLC) plates to determine the percentage of ATP hydrolyzed to ADP·Pi (21Sullivan C.S. Tremblay J.D. Fewell S.W. Lewis J.A. Brodsky J.L. Pipas J.M. Mol. Cell. Biol. 2000; 20: 5749-5757Crossref PubMed Scopus (74) Google Scholar, 45Fewell S.W. Day B.W. Brodsky J.L. J. Biol. Chem. 2001; 276: 910-914Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Data were obtained using different preparations of Hsp70. ATP turnover rates were calculated by using Kaleidagraph software (version 3.0.4) to fit the data to the single exponential equation,%Product=A×(1−exp(−kt))+C (Eq. 1) where the sum of the amplitude (A) represents the % of enzyme active sites that hydrolyze ATP to ADP·Pi, k is the rate of ATP hydrolysis in s-1, t is time in seconds, and the constant term (C) estimates the % of enzyme active sites that hydrolyze ATP to ADP·Pi at time 0 of the assay but also takes into account any contaminating ADP from the purchased isotope. Because ATP hydrolysis is rate-limiting for Hsp70s (7McCarty J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (349) Google Scholar), this experiment provides an approximation of the first-order rate constant for ATP hydrolysis. In assays to which co-chaperones were added, 1 μg of TAg or 0.5 μg of Ydj1p (final concentrations of ∼0.2 μm) was incubated with the [α32P]ATP-Hsp70 complex for 60 s at 30 °C before the addition of drug or Me2SO unless indicated otherwise. ATP hydrolysis rates were calculated using the equation described above from data obtained at the 60-s time point and beyond. However, it should be noted that because Ydj1p has been shown to increase the dissociation of ATP from purified ATP-Hsp70 complexes by ∼10-fold (55Ziegelhoffer T. Lopez-Buesa P. Craig E.A. J. Biol. Chem. 1995; 270: 10412-10419Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), a decrease in the amount of ATP hydrolysis in these reactions could result from inhibition of γ-phosphate cleavage and/or ATP rebinding. TAg ATPase Assays—Steady-state measurements of TAg ATPase activity were performed at 30 °C in 20-μl reactions containing 1 μg of TAg (0.56 μm) and 0.1 μCi of [α32P]ATP in 25 mm Hepes (pH 7.0), 5 mm MgCl2, 0.1 mm EDTA, 0.05% Nonidet P-40, 1 mm dithiothreitol, 25 μm ATP, and 2.5% (v/v) Me2SO or 0.3 mm MAL3-90 or MAL3-101. Aliquots were quenched and analyzed as described above for Hsp70 ATPase assays. Purified T antigen mutant 5031 was a kind gift from J. Pipas (University of Pittsburgh). In Vitro Translocation Assays—The synthesis and translocation of wild type and an unglycosylated form of yeast prepro-α-factor (ppαF and ΔG-ppαF, respectively) into yeast ER-derived vesicles were performed using published methods (45Fewell S.W. Day B.W. Brodsky J.L. J. Biol. Chem. 2001; 276: 910-914Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 57McCracken A.A. Brodsky J.L. J. Cell Biol. 1996; 132: 291-298Crossref PubMed Scopus (348) Google Scholar). Urea-denatured wild type substrate was synthesized and isolated as described (58Sanders S.L. Whitfield K.M. Vogel J.P. Rose M.D. Schekman R.W. Cell. 1992; 69: 353-365Abstract Full Text PDF PubMed Scopus (276) Google Scholar). Translocation reactions were preincubated with the indicated compounds or Me2SO for 10 min on ice before addition of an ATP-regenerating system. Translocation efficiency was determined by calculating the percent of trypsin-resistant, signal sequence-cleaved (and for wild type, glycosylated) pαF relative to the total amount of ppαF and pαF in reactions lacking trypsin. In Me2SO-containing control reactions, 20–30% of the input ppαF is typically converted to pαF. The relative translocation efficiencies were determined by dividing the translocation efficiency of the MAL3-39- and MAL3-101-containing reactions by the efficiency in the Me2SO-containing control reactions. Identification of Novel Modulators of Hsp70 ATPase Activity—Based upon their structural similarity to R/1 and DSG, we identified and obtained 31 small molecules (molecular weight range 314–1040; see Table I). Compound R/1 is comprised of two uracil (2,4-pyrimidinedione) ring systems connected by a linker containing two N-substituted carbamic ester moieties, each holding two hydrogen bond acceptors and one hydrogen bond donor (Fig. 1). In addition, the substitutions at position 5 on the uracil rings also contain three hydrogen bond acceptors and one donor. Therefore, we sought compounds that would follow similar but non-identical ring, linker, and H-bonding capacities. Several closely related compounds were found by sub-structural searching of the Developmental Therapeutics Program data base at the National Cancer Institute: NSC 625512, NSC 625513, NSC 625194, NSC 625195, NSC 624908, NSC 624905, NSC 624904, NSC 624393, NSC 624906, and NSC 624903. The NSC compounds selected have one or two uracil rings with substituents bearing hydrogen bond donors or acceptors. Compounds with the ML and MAL prefix were obtained from the University of Pittsburgh Center for Chemical Methodologies and Library Development and have 2-dihydropyrimidinone rings with differing substituents (see “Materials and Methods” for synthesis; Fig. 2A). These substituents contain amide links instead of carbamic ester links found in R/1. The amide systems also contain hydrogen bond donor or acceptor features. The effect of SC-ααδ9, a Cdc25 phosphatase inhibitor (59Rice R.L. Rusnak J.M. Yokokawa F. Yokokawa S. Messner D.J. Boynton A.L. Wipf P. Lazo J.S. Biochemistry. 1997; 36: 15965-15974Crossref PubMed Scopus (86) Google Scholar), was also examined, although there was no significant structural similarity with R/1 and DSG.Table IEffects of DSG and R/1 analogs on the rate of Hsp70 ATP hydrolysis ATPase assays were carried out as described under “Materials and Methods” with the indicated concentrations (0.1, 0.3, and 0.6 mm) of each compound. The rates of ATP hydrolysis were calculated using the formula % product = A × (1-exp(-kt)) + C, where the sum of the amplitude (A) represents the percentage of enzyme sites that hydrolyze ATP, k is the rate constant in s-1, t is time in seconds, and the constant term (C) estimates the percentage of enzyme sites that hydrolyze ATP at time 0. Rates shown are ×103 and are given as the percent ATP hydrolyzed to ADP·Pi/s from at least two independent experiments ± the range or S.D. ND, not determined. The relative “-Fold change” in the calculated rate of ATP hydrolysis in the presence of each compound compared to the rate of ATP hydrolysis in solvent (Me2SO) is indicated.Drug0.1 mm0.3 mm0.6 mmRate ATP hydrolysis-Fold changeRate ATP hydrolysis-Fold changeRate ATP hydrolysis-Fold changeMe2SO2.0 ± 0.1R/111.6 ± 1.05.8624392NDND1.6 ± 0.1-1.31.4 ± 0.2-1.4642393NDND1.2 ± 0.3-1.7NDND624903NDND0.9 ± 0.3-2.2NDND624904NDND1.4 ± 0.1-1.4NDND624905NDND1.7 ± 0.21.2NDND624906NDND1.1 ± 0.2-1.81.8 ± 0.7-1.1624907NDND2.4 ± 0.51.2NDND624908NDND1.8 ± 0.1-1.1NDND6251942.0 ± 0.213.6 ± 0.21.82.7 ± 0.41.46251956.1 ± 0.533.4 ± 0.31.72.5 ± 0.31.36255122.3 ± 0.21.26.2 ± 0.73.16.0 ± 0.536255132.2 ± 0.21.13.9 ± 0.324.9 ± 0.82.5632006NDND2.8 ± 0.31.41.0 ± 0.22655302NDND3.8 ± 0.51.91.5 ± 0.3-1.3aad9NDND1.2 ± 0.2-1.7NDNDML2-1933.2 ± 0.41.66.2 ± 0.53.14.5 ± 0.52.3ML2-1942.5 ± 0.31.34.1 ± 0.72.12.7 ± 0.11.4ML2-2141.8 ± 0.2-1.13.4 ± 0.51.71.6 ± 0.31.3ML2-2152.3 ± 0.11.24.3 ± 0.42.24.2 ± 1.42.1MAL3-383.5 ± 0.21.86.1 ± 0.83.14.1 ± 0.62MAL3-39NDND2.1 ± 0.411.4 ± 0.7-1.4MAL3-403.5 ± 0.31.85.4 ± 0.52.76.0 ± 0.73MAL3-51NDND1.7 ± 0.3-1.21.6 ± 0.2-1.3MAL3-533.7 ± 0.21.94.9 ± 0.62.59.1 ± 1.04.6MAL3-54NDND2.2 ± 0.31.12.5 ± 0.21.3MAL3-551.3 ± 0.1-1.52.8 ± 0.21.45.6 ± 0.92.8MAL3-873.8 ± 0.31.94.5 ± 0.62.35.2 ± 0.72.6MAL3-882.9 ± 0.31.53.1 ± 0.41.34.7 ± 0.72.4MAL3-904.1 ± 0.6210.3 ± 1.75.213.3 ± 1.66.7MAL3-913.6 ± 0.31.85.8 ± 1.22.99.9 ± 1.35MAL3-101NDND2.1 ± 0.312.4 ± 0.21.2 Open table in a new tab All 31 compounds described above were tested initially for their ability to modulate the endogenous ATPase activity of purified Ssa1p, a yeast Hsp70. Complexes of Hsp70 and [α-32P]ATP were formed on ice and purified from free [α-32P]ATP by gel filtration at 4 °C. The resulting [α32P]ATP-Hsp70 complexes were then incubated at 30 °C in the presence of the desired compound or solvent (Me2SO), and the conversion of Hsp70-bound ATP to ADP·Pi was monitored by thin layer chromatography (Fig. 3A). Because the radiolabeled ATP-Hsp70 complexes were purified from unbound nucleotide, this procedure estimates the rate of ATP hydrolysis and is more specific and sensitive than steady-state experiments that measure the full cycle of ATP binding, hydrolysis, and nucleotide exchange; however, the reader is referred to other studies in which these parameters were measured (see for example Refs. 7McCarty J.S. Buchberger A. Reinstein J. Bukau B. J. Mol. Biol. 1995; 249: 126-137Crossref PubMed Scopus (349) Google Scholar, 55Ziegelhoffer T. Lopez-Buesa P. Craig E.A. J. Biol. Chem. 1995; 270: 10412-10419Abstract" @default.
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• W2080409112 date "2004-12-01" @default.
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• W2080409112 title "Small Molecule Modulators of Endogenous and Co-chaperone-stimulated Hsp70 ATPase Activity" @default.
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• W2080409112 doi "https://doi.org/10.1074/jbc.m404857200" @default.
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