E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to dNDPs and is composed of two homodimeric subunits: R1 and R2. R1 binds NDPs and contains binding sites for allosteric effectors that control substrate specificity and turnover rate. R2 contains a diiron-tyrosyl radical (Y(*)) cofactor that initiates nucleotide reduction. Pre-steady-state experiments with wild type R1 or C754S/C759S-R1 and R2 were carried out to determine which step(s) are rate-limiting and whether both active sites of R1 can catalyze nucleotide reduction. Rapid chemical quench experiments monitoring dCDP formation gave k(obs) of 9 +/- 4 s(-1) with an amplitude of 1.7 +/- 0.4 equiv. This amplitude, generated in experiments with pre-reduced R1 (3 or 15 microM) in the absence of reductant, indicates that both monomers of R1 are active. Stopped-flow UV-vis spectroscopy monitoring the concentration of the Y(*) failed to reveal any changes from 2 ms to seconds under similar conditions. These pre-steady-state experiments, in conjunction with the steady-state turnover numbers for dCDP formation of 2-14 s(-1) at RNR concentrations of 0.05-0.4 microM (typical assay conditions), reveal that the rate-determining step is a physical step prior to rapid nucleotide reduction and rapid tyrosine reoxidation to Y(*). Steady-state experiments conducted at RNR concentrations of 3 and 15 microM, typical of pre-steady-state conditions, suggest that, in addition to the slow conformational change(s) prior to chemistry, re-reduction of the active site disulfide to dithiol or a conformational change accompanying this process can also be rate-limiting.
Heat shock protein 90 (Hsp90) is an emerging therapeutic target of interest for the treatment of cancer. Its role in protein homeostasis and the selective chaperoning of key signaling proteins in cancer survival and proliferation pathways has made it an attractive target of small molecule therapeutic intervention. 17-Allylamino-17-demethoxygeldanamycin (17-AAG), the most studied agent directed against Hsp90, suffers from poor physical-chemical properties that limit its clinical potential. Therefore, there exists a need for novel, patient-friendly Hsp90-directed agents for clinical investigation. IPI-504, the highly soluble hydroquinone hydrochloride derivative of 17-AAG, was synthesized as an Hsp90 inhibitor with favorable pharmaceutical properties. Its biochemical and biological activity was profiled in an Hsp90-binding assay, as well as in cancer-cell assays. Furthermore, the metabolic profile of IPI-504 was compared with that of 17-AAG, a geldanamycin analog currently in clinical trials. The anti-tumor activity of IPI-504 was tested as both a single agent as well as in combination with bortezomib in myeloma cell lines and in vivo xenograft models, and the retention of IPI-504 in tumor tissue was determined. In conclusion, IPI-504, a potent inhibitor of Hsp90, is efficacious in cellular and animal models of myeloma. It is synergistically efficacious with the proteasome inhibitor bortezomib and is preferentially retained in tumor tissues relative to plasma. Importantly, it was observed that IPI-504 interconverts with the known agent 17-AAG in vitro and in vivo via an oxidation-reduction equilibrium, and we demonstrate that IPI-504 is the slightly more potent inhibitor of Hsp90.T he heat shock response, first identified in 1962 by Ritossa (1), was initially characterized as the induction of select polypeptides in response to an acute cellular heat shock. These polypeptides were proteins that bound to partially unfolded proteins to prevent their aggregation and assist in their refolding (2, 3), and were termed chaperones. Of the heat shock proteins, heat shock protein 90 (Hsp90) in particular has been the subject of intense investigation. Work over the last decade has revealed not only a general protein chaperone role for Hsp90, but also a specific chaperone role in the binding of select conformations or metastable forms of signaling proteins (clients), thereby attenuating their signaling activity (4-6). Client proteins include the targets of key cancer survival and proliferation pathways, including Akt, Bcr-Abl, Her-2, mutant EGFR, and c-Kit, many of which are the subject of individual investigation for points of therapeutic intervention. Therefore, two functions of Hsp90 exist: (i) a general protein chaperone function (protein homeostasis) and (ii) a specific function to modulate the integrity of cell-signaling pathways through the proper folding of pathway members that are Hsp90 clients.Multiple myeloma (MM) is a neoplasm of terminally differentiated B cells (plasma cells) (7). Because of the high protein secr...
17-Allylamino-17-demethoxygeldanamycin (17-AAG)1 is a semisynthetic inhibitor of the 90 kDa heat shock protein (Hsp90) currently in clinical trials for the treatment of cancer. However, 17-AAG faces challenging formulation issues due to its poor solubility. Here we report the synthesis and evaluation of a highly soluble hydroquinone hydrochloride derivative of 17-AAG, 1a (IPI-504), and several of the physiological metabolites. These compounds show comparable binding affinity to human Hsp90 and its endoplasmic reticulum (ER) homologue, the 94 kDa glucose regulated protein (Grp94). Furthermore, the compounds inhibit the growth of the human cancer cell lines SKBR3 and SKOV3, which overexpress Hsp90 client protein Her2, and cause down-regulation of Her2 as well as induction of Hsp70 consistent with Hsp90 inhibition. There is a clear correlation between the measured binding affinity of the compounds and their cellular activities. Upon the basis of its potent activity against Hsp90 and a significant improvement in solubility, 1a is currently under evaluation in Phase I clinical trials for cancer.
Escherichia coli class I ribonucleotide reductase catalyzes the conversion of ribonucleotides to deoxyribonucleotides and consists of two subunits: R1 and R2. R1 possesses the active site, while R2 harbors the essential diferric-tyrosyl radical (Y*) cofactor. The Y* on R2 is proposed to generate a transient thiyl radical on R1, 35 A distant, through amino acid radical intermediates. To study the putative long-range proton-coupled electron transfer (PCET), R2 (375 residues) was prepared semisynthetically using intein technology. Y356, a putative intermediate in the pathway, was replaced with 2,3-difluorotyrosine (F2Y, pKa = 7.8). pH rate profiles (pH 6.5-9.0) of wild-type and F2Y-R2 were very similar. Thus, a proton can be lost from the putative PCET pathway without affecting nucleotide reduction. The current model involving H* transfer is thus unlikely.
High-frequency pulsed EPR and ENDOR have been employed to characterize the tyrosyl radical (Y*)-diiron cofactor in the Y2-containing R2 subunit of ribonucleotide reductase (RNR) from yeast. The present work represents the first use of 140-GHz time domain EPR and ENDOR to examine this system and demonstrates the capabilities of the method to elucidate the electronic structure and the chemical environment of protein radicals. Low-temperature spin-echo-detected EPR spectra of yeast Y* reveal an EPR line shape typical of a tyrosyl radical; however, when compared with the EPR spectra of Y* from E. coli RNR, a substantial upfield shift of the g(1)-value is observed. The origin of the shift in g(1) was investigated by 140-GHz (1)H and (2)H pulsed ENDOR experiments of the Y2-containing subunit in protonated and D(2)O-exchanged buffer. (2)H ENDOR spectra and simulations provide unambiguous evidence for one strongly coupled (2)H arising from a bond between the radical and an exchangeable proton of an adjacent residue or a water molecule. Orientation-selective 140-GHz ENDOR spectra indicate the direction of the hydrogen bond with respect to the molecular symmetry axes and the bond length (1.81 A). Finally, we have performed saturation recovery experiments and observed enhanced spin lattice relaxation rates of the Y* above 10 K. At temperatures higher than 20 K, the relaxation rates are isotropic across the EPR line, a phenomenon that we attribute to isotropic exchange interaction between Y* and the first excited paramagnetic state of the diiron cluster adjacent to it. From the activation energy of the rates, we determine the exchange interaction between the two irons of the cluster, J(exc) = -85 cm(-)(1). The relaxation mechanism and the presence of the hydrogen bond are discussed in terms of the differences in the structure of the Y*-diiron cofactor in yeast Y2 and other class I R2s.
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides. Class I RNRs are composed of two homodimeric subunits: R1 and R2. R1 is directly involved in the reduction, and R2 contains the diferric-tyrosyl radical (Y⅐) cofactor essential for the initiation of reduction. Saccharomyces cerevisiae has two RNRs; Y1 and Y3 correspond to R1, whereas Y2 and Y4 correspond to R2. Y4 is essential for diferric-Y⅐ formation in Y2 from apoY2, Fe 2؉ , and O2. The actual function of Y4 is controversial. Y2 and Y4 have been further characterized in an effort to understand their respective roles in nucleotide reduction. (His) 6-Y2, Y4, and (His) 6-Y4 are homodimers, isolated largely in apo form. Their CD spectra reveal that they are predominantly helical. The concentrations of Y2 and Y4 in vivo are 0. R ibonucleotide reductases (RNRs) play an essential role in DNA replication and repair by providing all of the monomeric deoxynucleotides required for these processes. The genome sequencing projects have revealed that both prokaryotes and eukaryotes possess multiple RNRs, whose functions remain to be elucidated (1). Class I RNRs are composed of two homodimeric subunits: R1 (␣2) and R2 (2). Both subunits are essential for activity. R1 contains the binding sites for the nucleoside diphosphate substrates and the deoxynucleotide and ATP allosteric effectors that govern which nucleotide is reduced and its rate of reduction. R2 possesses a diferric-tyrosyl radical (Y⅐) cofactor essential for initiation of nucleotide reduction on R1 (2, 3).Four genes have been identified that encode RNR subunits in Saccharomyces cerevisiae: RNR1 and RNR3 (their gene products designated Y1 and Y3) are R1 homologues, and RNR2 and RNR4 (their gene products designated Y2 and Y4) are R2 homologues (4-9). Y1 and Y3 share 80% sequence identity (4). RNR1 expression is cell cycle regulated, and the gene is essential for mitotic viability (5, 10). In contrast, RNR3 is not expressed under normal growth conditions. Transcription of RNR1 and RNR3 is inducible by DNA damage; the mRNA of the latter is up-regulated Ͼ500-fold (5). Y2 and Y4 share 56% sequence identity. Y4 contains several unusual features relative to a canonical R2. It lacks about 50 amino acid residues from the N terminus, and of the 16 amino acid residues conserved in almost all class I R2s, six have been replaced in Y4. The most notable substitutions are two histidines and a glutamate, ligands of the di-iron center, which have been replaced by two tyrosines and an arginine. Such substitutions would be expected to disrupt the ability of Y4 to bind iron. RNR2 is essential for mitotic viability (10,11). RNR4 also appears to be important for mitotic viability, but its essentiality varies with genetic background (8, 9, 12). In the one case where a Y4 knockout was shown to be lethal, the lethality could be suppressed by overexpression of Y1 and Y3 (8).The transcriptional regulation of the yeast RNR genes has been studied extensively. However, little is known about the biochem...
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